RESUMO
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
Background: Current treatments for progressive neurodegenerative disorders characterized by cognitive impairment either have limited efficacy or are lacking altogether. SDI-118 is a small molecule which modulates the activity of synaptic vesicle glycoprotein 2A (SV2A) in the brain and shows cognitive enhancing effects in a range of animal models of cognitive deficit. Methods: This first-in-human study evaluated safety, tolerability, and pharmacokinetics/pharmacodynamics of SDI-118 in single ascending oral doses up to 80 mg administered to 32 healthy male subjects. Brain target occupancy was measured in eight subjects using positron emission tomography with PET-ligand [11C]-UCB-J. Food effect was assessed in seven subjects. Mood state was regularly evaluated using standardized questionnaires, and resting state fMRI data were analyzed as exploratory objectives. Key Results: At all doses tested, SDI-118 was well tolerated and appeared safe. Adverse events were mainly dizziness, hypersomnia, and somnolence. All were mild in intensity and increased in frequency with increasing administered dose. No dose-limiting adverse reactions were observed at any dose. SDI-118 displayed a linear pharmacokinetic profile with no significant food effect. Brain penetration and target engagement were demonstrated by a dose-proportional SV2A occupancy. Conclusion: Single oral doses of SDI-118 up to 80 mg were very well tolerated in healthy male subjects. Dose-proportional SV2A occupancy in the brain was demonstrated with brain imaging. Adverse effects in humans mainly occurred in higher dose ranges, with high occupancy levels, and were all mild and self-limiting. These data support further clinical exploration of the compound in patients with cognitive disorders. Clinical Trial Registration: https://clinicaltrials.gov/, identifier NCT05486195.
RESUMO
Since the mid 1980s, there has been a great deal of enthusiasm within both academia and industry about the therapeutic potential of drugs targeting the NMDA subtype of glutamate receptors. That early promise is just beginning to translate into approvable drugs. Here we review the reasons for this slow progress and critically assess the future prospects for drugs that act on NMDA receptor pathways, including potential treatments for some major disorders such as stroke and Alzheimer's disease, for which effective therapies are still lacking.
Assuntos
Encefalopatias/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Desenho de Fármacos , Indústria Farmacêutica/tendências , Vias Neurais/efeitos dos fármacos , Neurociências/tendências , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Animais , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Encefalopatias/metabolismo , Encefalopatias/fisiopatologia , Agonistas de Aminoácidos Excitatórios/uso terapêutico , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Humanos , Vias Neurais/metabolismo , Vias Neurais/fisiopatologia , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
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
Glutamate- and GABA-releasing neurons form the basis for neurotransmission in the mammalian central nervous system (CNS). The co-ordination of these excitatory and inhibitory systems, together with intrinsic voltage-gated ion channels and G-protein-coupled receptor modulation, provides the diverse neuronal firing patterns, network activity and synaptic plasticity that are required for the complexity of CNS function. Virtually all of the known molecular components of the gamma-aminobutyric acid (GABA) and glutamate neurotransmitter systems have been considered as potential therapeutic targets. Positive allosteric modulators of GABAA receptors, such as the benzodiazepines, have found wide clinical use, and the N-methyl-D-aspartate receptor antagonists ketamine and memantine have therapeutic utility. In these fundamental neurotransmitter systems, drugs that provide allosteric modulation of ligand-gated ion channels or G-protein-coupled receptors, or seek to selectively target receptor subtypes, appear to hold the greatest promise for the desired balance of efficacy and tolerability. This might also be achieved through targeting transporter subtypes. A large number of compounds based on these strategies are currently in clinical trials for diseases that span a wide range of CNS disorders.
Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Agonistas GABAérgicos/farmacologia , Antagonistas GABAérgicos/farmacologia , Animais , Sistema Nervoso Central/metabolismo , Ensaios Clínicos como Assunto , Agonistas de Aminoácidos Excitatórios/uso terapêutico , Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Agonistas GABAérgicos/uso terapêutico , Antagonistas GABAérgicos/uso terapêutico , Ácido Glutâmico/metabolismo , Humanos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Receptores de AMPA/efeitos dos fármacos , Receptores de AMPA/metabolismo , Receptores de GABA/efeitos dos fármacos , Receptores de GABA/metabolismo , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Synaptic dysfunction is a well-documented manifestation in animal models of Alzheimer's disease pathology. In this context, numerous studies have documented reduction in the functionality of synapses in various models. In addition, recent research has shed more light on increased excitability and its link to seizures and seizure-like activities in AD patients as well as in mouse models. These reports of hyperexcitability contradict the observed reduction in synaptic function and have been suggested to be as a result of the interplay between inhibitory and excitatory neuronal mechanism. The present study therefore investigates functional deficiency in the inhibitory system as complementary to the identified alterations in the glutamate excitatory pathway in AD. Since synaptic function deficit in AD is typically linked to progression/pathology of the disease, it is important to determine whether the deficits in the GABAergic system are functional and can be directly linked to the pattern of the disruption documented in the glutamate system. To build on previous research in this field, experiments were designed to determine if previously documented synaptic dysfunction in AD models is concomitantly observed with excitation/inhibition imbalance as suggested by observation of seizure and seizure-like pathology in such models. We report changes in synaptic function in aged APPPS1 mice not observable in the younger cohort. These changes in synaptic function are furthermore accompanied by alteration in the GABAergic neurotransmission. Thus, age-dependent alteration in the inhibitory/excitatory balance might underpin the symptomatic changes observed with the progression of Alzheimer's disease pathology including sleep disturbance and epileptic events.
Assuntos
Envelhecimento , Doença de Alzheimer/patologia , Hipocampo/patologia , Potenciais Pós-Sinápticos Inibidores/genética , Sinapses/fisiologia , Ácido gama-Aminobutírico/metabolismo , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Estimulação Elétrica , GABAérgicos/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiopatologia , Humanos , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Presenilina-1/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sinapses/efeitos dos fármacosRESUMO
NMDA receptor hypofunction has been implicated in the pathophysiology of schizophrenia, and pharmacological and genetic approaches have been used to model such dysfunction. We previously have described two mouse lines carrying point mutations in the NMDA receptor glycine binding site, Grin1(D481N) and Grin1(K483Q), which exhibit 5- and 86-fold reductions in receptor glycine affinity, respectively. Grin1(D481N) animals exhibit a relatively mild phenotype compatible with a moderate reduction in NMDA receptor function, whereas Grin1(K483Q) animals die shortly after birth. In this study we have characterized compound heterozygote Grin1(D481N/K483Q) mice, which are viable and exhibited biphasic NMDA receptor glycine affinities compatible with the presence of each of the two mutated alleles. Grin1(D481N/K483Q) mice exhibited a marked NMDA receptor hypofunction revealed by deficits in hippocampal long-term potentiation, which were rescued by the glycine site agonist d-serine, which also facilitated NMDA synaptic currents in mutant, but not in wild-type, mice. Analysis of striatal monoamine levels revealed an apparent dopaminergic and serotonergic hyperfunction. Behaviorally, Grin1(D481N/K483Q) mice were insensitive to acute dizocilpine pretreatment and exhibited increased startle response but normal prepulse inhibition. Most strikingly, mutant mice exhibited a sustained, nonhabituating hyperactivity and increased stereotyped behavior that were resistant to suppression by antipsychotics and the benzodiazepine site agonist Zolpidem. They also displayed a disruption of nest building behavior and were unable to perform a cued learning paradigm in the Morris water maze. We speculate that the severity of NMDA receptor hypofunction in these mice may account for their profound behavioral phenotype and insensitivity to antipsychotics.
Assuntos
Resistência a Medicamentos/genética , Glicina/metabolismo , Hipercinese/genética , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Antipsicóticos/farmacologia , Comportamento Animal/efeitos dos fármacos , Sítios de Ligação/genética , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/genética , Aminas Biogênicas/metabolismo , Corpo Estriado/química , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Relação Dose-Resposta a Droga , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Agonistas GABAérgicos/farmacologia , Marcação de Genes , Ácido Glutâmico/farmacocinética , Glicina/agonistas , Glicina/farmacocinética , Heterozigoto , Hipocampo/fisiopatologia , Hipercinese/fisiopatologia , Técnicas In Vitro , Potenciação de Longa Duração/genética , Camundongos , Camundongos Mutantes Neurológicos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Técnicas de Patch-Clamp , Fenótipo , Mutação Puntual , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Serina/análogos & derivados , Serina/farmacologia , Estereoisomerismo , Comportamento Estereotipado/efeitos dos fármacosRESUMO
Transgenic mice, expressing mutant beta-amyloid precursor proteins (betaAPPs), have lead to a better understanding of the pathophysiological processes in Alzheimer's disease (AD). In many of these models, however, the temporal development of cognitive decline and the relationship to Abeta deposition and inflammation are unclear. We now report a novel transgenic mouse line, PS2APP (PS2N141I x APPswe), which develops a severe cerebral amyloidosis in discrete brain regions, and present a cross-sectional analysis of these mice at 4, 8, 12, and 16 months of age. Each age cohort was investigated for changes in behavior, electrophysiology of synapse efficacy, ELISA-determined Abeta load, histopathology, and in immunoelectron microscopy. Cognitive deficits were first observed at 8 months when Abeta deposits and inflammation were restricted to discrete brain regions, namely the subiculum and frontolateral (motor and orbital) cortex. As early as 5 months, electron microscopy revealed the presence, in these regions, of pre-plaque, immunogold-labeled extracellular fibrillar Abeta. At the same age, increased levels of insoluble Abeta were detected by ELISA, with Abeta1-40 levels exceeding those of Abeta1-42. Further cognitive decline occurred in an age-related manner, and this was accompanied by the spread of amyloidosis to ultimately affect not only neo- and limbic cortices, but also thalamic and pontine nuclei. Dentate gyrus post-tetanic potentiation was significantly attenuated at 17 months, and there were also significant differences in paired-pulse parameters. This systematic cross-sectional study of the behavioral and pathological changes in the PS2APP mouse indicates that it develops age-related cognitive decline associated with severe amyloidosis and inflammation in discrete brain regions and therefore is suitable for testing a range of potential symptomatic and disease-modifying therapies for AD.
Assuntos
Precursor de Proteína beta-Amiloide/biossíntese , Amiloidose/fisiopatologia , Encéfalo/metabolismo , Transtornos Cognitivos/fisiopatologia , Proteínas de Membrana/biossíntese , Fatores Etários , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/genética , Amiloidose/complicações , Amiloidose/patologia , Animais , Comportamento Animal , Encéfalo/patologia , Química Encefálica , Transtornos Cognitivos/complicações , Transtornos Cognitivos/patologia , Estudos Transversais , Cruzamentos Genéticos , Modelos Animais de Doenças , Progressão da Doença , Hipocampo/fisiopatologia , Humanos , Técnicas In Vitro , Masculino , Aprendizagem em Labirinto , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Microscopia Imunoeletrônica , Mutação , Plasticidade Neuronal/genética , Presenilina-2 , Transmissão Sináptica/genéticaRESUMO
RATIONALE: L: -Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS) and mediates its actions via activation of both ionotropic and metabotropic receptor families. The development of selective ligands, including competitive agonists and antagonists and positive and negative allosteric modulators, has enabled investigation of the functional roles of glutamate receptor family members. OBJECTIVE: In this review we describe the subunit structure and composition of the ionotropic and metabotropic glutamate receptors and discuss their pharmacology, particularly with respect to selective tools useful for investigation of their function in the CNS. RESULTS: A large number of ligands are now available that are selective either for glutamate receptor subfamilies or for particular receptor subtypes. Such ligands have enabled considerable advances in the elucidation of the physiological and pathophysiological roles of receptor family members. Furthermore, efficacy in animal models of neurological and psychiatric disorders has supported the progression of several glutamatergic ligands into clinical studies. These include ionotropic glutamate receptor antagonists, which have entered clinical trials for disorders including epilepsy and ischaemic stroke, alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) receptor positive allosteric modulators which are under evaluation as cognitive enhancers, and metabotropic glutamate receptor 2 (mGluR2) agonists which are undergoing clinical evaluation as anxiolytics. Furthermore, preclinical studies have illustrated therapeutic potential for ligands selective for other receptor subtypes in various disorders. These include mGluR1 antagonists in pain, mGluR5 antagonists in anxiety, pain and drug abuse and mGluR5 positive allosteric modulators in schizophrenia. CONCLUSIONS: Selective pharmacological tools have enabled the study of glutamate receptors. However, pharmacological coverage of the family is incomplete and considerable scope remains for the development of novel ligands, particularly those with in vivo utility, and for the their use together with existing tools for the further investigation of the roles of receptor family members in CNS function and as potentially novel therapeutics.
Assuntos
Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Receptores de AMPA/química , Receptores de Ácido Caínico/química , Receptores de Glutamato Metabotrópico/química , Receptores de N-Metil-D-Aspartato/química , Regulação Alostérica , Animais , Ligação Competitiva , Humanos , Ligantes , Receptores de AMPA/efeitos dos fármacos , Receptores de Ácido Caínico/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacosRESUMO
RATIONALE: We previously reported that the NR2B subunit-selective N-methyl-D-aspartate (NMDA) antagonist Ro 63-1908 produced a marked deficit in response control in the five-choice serial reaction time task (5-CSRTT). OBJECTIVES: The present studies were designed to investigate this further by studying the NR2B NMDA antagonists, ifenprodil, traxoprodil (CP101,606), Ro 25-6981 as well as Ro 63-1908 in this test. METHODS: Following training in the 5-CSRTT, separate groups of rats were either tested under (1) standard test conditions [5 s inter-trial interval (ITI), 0.5 s stimulus duration, 100 trials], (2) high (3 s ITI) and low (10 s ITI) event rate of stimulus presentation and (3) a 250-trial protocol in aged 2-year-old rats. In a final study, the effects of traxoprodil were investigated in an operant delayed match to position (DMTP) task, a test of working memory, and compared to dizocilpine and Ro 63-1908. RESULTS: Similar to Ro 63-1908, both traxoprodil (1-10 mg/kg) and Ro 25-6981 (3--30 mg/kg) increased premature responding but also increased response speed with no error trade-off. Conversely, ifenprodil (1--10 mg/kg) slowed response speed and increased omissions with no effect on premature responding. Tested under a variable ITI, Ro 63--1908 (1 mg/kg) increased premature responding at all ITIs, but this change was proportional to controls. At short ITI (3 s), Ro 63-1908 reliably improved performance both in terms of response speed and accuracy (percent correct). In a 250-trial protocol in aged rats, both Ro 63-1908 (0.1-0.3 mg/kg) and, particularly, traxoprodil (1--3 mg/kg) improved performance-increasing response speed and increasing the number of rewards earned during test. Finally, traxoprodil (1--10 mg/kg) improved accuracy and increased response speed in the DMTP task. CONCLUSIONS: The present studies support the view that selective NR2B NMDA antagonists promote impulsive-type responding in the 5-CSRTT; however, under certain test conditions, drugs of this class-notably traxoprodil-may also improve task performance.
Assuntos
Cognição/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Maleato de Dizocilpina/farmacologia , Masculino , Fenóis/farmacologia , Piperidinas/farmacologia , Desempenho Psicomotor/efeitos dos fármacos , Ratos , Tempo de Reação/efeitos dos fármacosRESUMO
Human C-reactive protein (CRP), the classic acute phase plasma protein, increases in concentration after myocardial infarction and stroke. Human CRP binds to ligands exposed in damaged tissue and can then activate complement and its proinflammatory functions. In contrast, rat CRP, which binds to similar ligands, does not activate complement. In the present study, systemic complement depletion with cobra venom factor in adult rats subjected to middle cerebral artery occlusion did not affect cerebral infarct size, indicating that circulating complement does not contribute to injury in this model. However, we have previously reported that administration of human CRP to rats undergoing coronary artery ligation caused a marked increase in size of the resulting myocardial infarction, associated with codeposition of human CRP and rat complement in the infarcts. In the present study, we show that adult rats subjected to middle cerebral artery occlusion and then treated with human CRP similarly developed significantly larger cerebral infarcts compared with control subjects receiving human serum albumin. Human CRP can thus contribute to ischemic tissue damage in the brain as well as in the heart, and inhibition of CRP binding may therefore be a promising target for tissue protective acute therapeutic intervention in stroke as well as in myocardial infarction.
Assuntos
Envelhecimento/fisiologia , Proteína C-Reativa/toxicidade , Infarto da Artéria Cerebral Média/induzido quimicamente , Infarto da Artéria Cerebral Média/patologia , Animais , Humanos , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
These studies have addressed the role of caspase-3 activation in neuronal death after cerebral ischemia in different animal models. The authors were unable to show activation of procaspase-3 measured as an induction of DEVDase (Asp-Glu-Val-Asp) activity after focal or transient forebrain ischemia in rats. DEVDase activity could not be induced in the cytosolic fraction of the brain tissue obtained from these animals by exogenous cytochrome c/dATP and Ca2+. However, the addition of granzyme B to these cytosolic fractions resulted in a significant activation of DEVDase, confirming that the conditions were permissive to analyze proteolytic cleavage of the DEVD-AMC (7-amino-4-methyl-coumarin) substrate. Consistent with these findings, zVal-Ala-Asp-fluoromethylketone administered after focal ischemia did not have a neuroprotective effect. In contrast to these findings, a large increase in DEVDase activity was detected in a model of hypoxic-ischemia in postnatal-day-7 rats. Furthermore, in postnatal-day-7 animals treated with MK-801, in which it has been suggested that excessive apoptosis is induced, the authors were unable to detect activation of DEVDase activity but were able to induce it in vitro by the addition of cytochrome c/dATP and Ca2+ to the cytosolic fraction. Analysis of cytochrome c distribution did not provide definitive evidence for selective cytochrome c release in the permanent focal ischemia model, whereas in the transient model a small but consistent amount of cytochrome c was found in the cytosolic fraction. However, in both models the majority of cytochrome c remained associated with the mitochondrial fraction. In conclusion, the authors were unable to substantiate a role of mitochondrially derived cytochrome c and procaspase-3 activation in ischemia-induced cell death in adult brain, but did see a clear induction of caspase-3 in neonatal hypoxia.
Assuntos
Isquemia Encefálica/fisiopatologia , Encéfalo/enzimologia , Caspases/metabolismo , Neurônios/fisiologia , Peptídeo Hidrolases/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Animais Recém-Nascidos , Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Encéfalo/fisiopatologia , Isquemia Encefálica/enzimologia , Isquemia Encefálica/patologia , Caspase 3 , Morte Celular , Grupo dos Citocromos c/metabolismo , Maleato de Dizocilpina/farmacologia , Ativação Enzimática , Granzimas , Humanos , Infarto da Artéria Cerebral Média , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Fármacos Neuroprotetores/farmacologia , Precursores de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Serina Endopeptidases/farmacologiaRESUMO
Group II metabotropic glutamate (mGlu) receptors can act as presynaptic autoinhibitory receptors at perforant path inputs to the hippocampus under conditions of high frequency synaptic activation. We have used mGlu2 -/- mice to examine the relative roles of mGlu2 and mGlu3 in the regulation of perforant path synaptic transmission mediated by both the selective group II receptor agonist, DCG-IV, and by synaptically released glutamate. Field excitatory postsynaptic potentials evoked by stimulation of either the perforant path inputs to the dentate gyrus mid-moleculare or the CA1 stratum lacunosum moleculare were inhibited by DCG-IV with IC(50) values and maximum percentage inhibition of: 169 nM (60%) and 41 nM (72%) in wild-type mice and 273 nM (19%) and 116 nM (49%) in mGlu2 -/- mice, respectively. Activation of presynaptic group II mGlu autoreceptors by synaptically released glutamate, as revealed by a LY341495-mediated increase in the relative amplitude of a test fEPSP evoked after a conditioning burst, was observed in both the dentate gyrus and the stratum lacunosum of wild-type, but not mGlu2 -/- mice. These observations demonstrate that activation of mGlu3 receptors can regulate synaptic transmission at perforant path synapses but suggest that mGlu2 is the major presynaptic group II autoreceptor activated by synaptically released glutamate.
Assuntos
Giro Denteado/fisiologia , Via Perfurante/fisiologia , Receptores de Glutamato Metabotrópico/deficiência , Receptores de Glutamato Metabotrópico/fisiologia , Transmissão Sináptica/fisiologia , Animais , Giro Denteado/efeitos dos fármacos , Relação Dose-Resposta a Droga , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Camundongos , Via Perfurante/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/genética , Transmissão Sináptica/efeitos dos fármacosRESUMO
Atrophy of the medial temporal lobes, including the glutamatergic cortical-hippocampal circuitry, is an early event in Alzheimer's disease (AD) and probably contributes to the characteristic short-term mnemonic decline. Pharmacological strategies directly targeted to ameliorating this functional decline may represent a novel approach for the symptomatic treatment of AD. Presynaptic group II metabotropic glutamate receptors (i.e. mGlu2 and mGlu3) exert a powerful modulatory influence on the function of these pathways, in particular the perforant pathway. Using a combination of mGlu2 receptor knockout mice and the group II agonist LY354740, we show that activation of mGlu2 receptors produces a cognitive impairment, i.e. a delay-dependent deficit in delayed matching and non-matching to position, and impaired spatial learning in a Morris water maze. Conversely, a group II antagonist, LY341495, improved acquisition of spatial learning. LY354740 potently reduced field excitatory postsynaptic potentials in hippocampal slices from wild type but not mGlu2 receptor knockout mice. Taken together, these results suggest that activation of mGlu2 receptors evokes a powerful inhibitory effect on hippocampal synaptic transmission and mGlu2 agonists produce a cognitive deficit consistent with this change. Conversely, mGlu2 receptor antagonists may improve certain aspects of cognition and thus represent a novel approach for the symptomatic treatment of AD.
Assuntos
Cognição/efeitos dos fármacos , Desempenho Psicomotor/efeitos dos fármacos , Tempo de Reação/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Aminoácidos/farmacologia , Animais , Compostos Bicíclicos com Pontes/farmacologia , Cognição/fisiologia , Relação Dose-Resposta a Droga , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Desempenho Psicomotor/fisiologia , Ratos , Tempo de Reação/fisiologia , Receptores de Glutamato Metabotrópico/fisiologia , Xantenos/farmacologiaRESUMO
The G-protein coupled metabotropic glutamate receptor mGlu5 plays a pivotal role as a modulator of synaptic plasticity, ion channel activity and excitotoxicity. Two splice variants, hmGlu5a and -5b have been reported previously. During screening of a human brain cDNA library for hmGlu5a, we identified a novel variant (hmGlu5d) generated by alternative splicing at the C-terminal domain. The predicted hmGlu5d protein has a C-terminal 267 amino acid shorter than that of hmGlu5a. The pattern of mRNA expression of mGluR5 variants in human brain were analyzed by RT-PCR and in situ hybridization histochemistry. RT-PCR analysis demonstrated the presence of the hmGlu5d transcript, although at low level, in human whole brain, cerebellum, cerebral cortex and hippocampus. [3H]Quisqualate displayed similar affinity at the hmGlu5 splice variants (K(D) values of 80+/-8 and 54+/-17 nM for hmGlu5a and -5d receptors, respectively). For the five mGlu agonists studied, a similar rank order of potency was observed on both hmGlu5a and -5d receptors: quisqualate>glutamate>DHPG>L-CCGI approximately ACPD. MPEP inhibited the glutamate (2 microM)-induced [Ca(2+)](i) response in hmGlu5a and -5d-HEK293 cells also with similar potency (IC(50) values 25+/-1.5 and 20+/-1.4 nM, respectively). Therefore, the large truncation of the C-terminal tail of mGlu5 does not have any apparent major effect on the potency and efficacy of agonists as measured by the [Ca(2+)](i) responses or by activation of recombinant G-protein coupled inwardly rectifying K(+) (GIRK) channel currents. The only major functional difference is the increased sensitivity of hmGlu5d to protein kinase C (PKC)-mediated desensitization, relative to hmGlu5a.
Assuntos
Processamento Alternativo , Cerebelo/fisiologia , Hipocampo/fisiologia , Receptores de Glutamato Metabotrópico/genética , Idoso , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Linhagem Celular , Cerebelo/citologia , Cricetinae , Relação Dose-Resposta a Droga , Agonistas de Aminoácidos Excitatórios/metabolismo , Feminino , Ácido Glutâmico/metabolismo , Hipocampo/citologia , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ácido Quisquálico/metabolismo , Receptor de Glutamato Metabotrópico 5 , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/metabolismo , Alinhamento de SequênciaRESUMO
RATIONALE: Glutamate signalling through the N-methyl-D-aspartate (NMDA) receptor is of critical importance for normal central nervous system (CNS) function, as indicated by the marked behavioural disturbances produced by non-subtype selective NMDA antagonists such as dizocilpine (MK-801). OBJECTIVE: The present studies were designed to investigate the involvement of the two major NMDA receptor subunits in the central nervous system, i.e. NR2A and NR2B, on sensorimotor gating in mice. METHODS: These experiments utilised the non-subtype-selective NMDA antagonist dizocilpine, a line of NR2A-KO mice and the selective NR2B antagonist Ro 63-1908, in the study of pre-pulse inhibition of the startle response (PPI). RESULTS: The non-selective NMDA receptor antagonist dizocilpine (0.1-1 mg/kg, IP) robustly disrupted PPI in wild-type mice. Conversely, selective genetic or pharmacological inhibition of either the NMDA NR2A or NR2B receptor subunit containing receptors, respectively, had no effect on PPI. Thus, NR2A KO mice showed normal PPI compared with wild-type littermate controls, and administration of Ro 63-1908 (1-10 mg/kg IP) to wild-type mice did not affect PPI. However, selective inhibition of NR2A and NR2B by administration of Ro 63-1908 to NR2A KO mice significantly disrupted PPI. CONCLUSIONS: These data imply that concomitant inhibition of both NR2A and NR2B subunit-containing NMDA receptors is necessary to disrupt PPI, suggesting that inhibition of NR2A and NR2B-containing NMDA receptors is required to elicit behaviours suggestive of psychomimetic effects in man.
Assuntos
Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/genética , Reflexo de Sobressalto/efeitos dos fármacos , Reflexo de Sobressalto/genética , Animais , Maleato de Dizocilpina/administração & dosagem , Maleato de Dizocilpina/farmacologia , Relação Dose-Resposta a Droga , Antagonistas de Aminoácidos Excitatórios/administração & dosagem , Antagonistas de Aminoácidos Excitatórios/farmacologia , Genótipo , Masculino , Camundongos , Camundongos Knockout , Fenóis/administração & dosagem , Fenóis/farmacologia , Piperidinas/administração & dosagem , Piperidinas/farmacologia , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genéticaRESUMO
BACKGROUND: Transgenic mice expressing mutated amyloid precursor protein (APP) and presenilin (PS)-1 or -2 have been successfully used to model cerebral beta-amyloidosis, one of the characteristic hallmarks of Alzheimer's disease (AD) pathology. However, the use of many transgenic lines is limited by premature death, low breeding efficiencies and late onset and high inter-animal variability of the pathology, creating a need for improved animal models. Here we describe the detailed characterization of a new homozygous double-transgenic mouse line that addresses most of these issues. METHODOLOGY/PRINCIPAL FINDINGS: The transgenic mouse line (ARTE10) was generated by co-integration of two transgenes carrying the K670N/M671L mutated amyloid precursor protein (APP(swe)) and the M146V mutated presenilin 1 (PS1) both under control of a neuron-specific promoter. Mice, hemi- as well as homozygous for both transgenes, are viable and fertile with good breeding capabilities and a low rate of premature death. They develop robust AD-like cerebral beta-amyloid plaque pathology with glial inflammation, signs of neuritic dystrophy and cerebral amyloid angiopathy. Using our novel image analysis algorithm for semi-automatic quantification of plaque burden, we demonstrate an early onset and progressive plaque deposition starting at 3 months of age in homozygous mice with low inter-animal variability and 100%-penetrance of the phenotype. The plaques are readily detected in vivo by PiB, the standard human PET tracer for AD. In addition, ARTE10 mice display early loss of synaptic markers and age-related cognitive deficits. By applying a gamma-secretase inhibitor we show a dose dependent reduction of soluble amyloid beta levels in the brain. CONCLUSIONS: ARTE10 mice develop a cerebral beta-amyloidosis closely resembling the beta-amyloid-related aspects of human AD neuropathology. Unifying several advantages of previous transgenic models, this line particularly qualifies for the use in target validation and for evaluating potential diagnostic or therapeutic agents targeting the amyloid pathology of AD.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Amiloidose/genética , Animais , Modelos Animais de Doenças , Feminino , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Neurônios/metabolismo , Presenilina-1/genética , Regiões Promotoras GenéticasRESUMO
The asymmetric synthesis and receptor pharmacology of (1S,2R,3R,5R,6S)-2-amino-3-Hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid (+)-9 (HYDIA) and a few of its O-alkylated derivatives are described. The key step of the synthesis utilizes Sharpless' asymmetric dihydroxylation (AD-beta) for the kinetic resolution of a bicyclic racemic precursor olefin. In contrast to the bicyclic glutamate analogue LY354740, which is a potent and selective agonist for the group II metabotropic glutamate receptors (mGluRs), these new conformationally restricted and also hydroxylated or alkoxylated glutamate analogues are potent and selective antagonists for the group II mGluRs.
Assuntos
Compostos Bicíclicos com Pontes/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Receptores de Glutamato Metabotrópico/agonistas , Alquilação , Animais , Ligação Competitiva , Compostos Bicíclicos com Pontes/síntese química , Agonistas de Aminoácidos Excitatórios/síntese química , Ácido Glutâmico/química , Ácido Glutâmico/farmacologia , Hidroxilação , Ligantes , Camundongos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
N-methyl-D-aspartate (NMDA) receptor antagonists have antiakinetic and antidyskinetic effects in animals models of Parkinson's disease (PD). However, non-selective inhibition of NMDA receptors throughout the central nervous system may result in undesired effects such as ataxia and psychosis. We therefore studied Ro 25-6981, an activity-dependent antagonist of NMDA receptors containing the NR2B subunit which are predominantly expressed in the striatum. Ro 25-6981 induced contraversive rotations in 6-hydroxydopamine (6-OHDA)-lesioned rats without stimulating locomotion in normal rats and reversed parkinsonian symptoms in 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP)-treated common marmosets. Due to the small number of marmosets, there were no significant differences between Ro 25-6981 and vehicle though there was a significant trend toward differences, as shown by the Page test. Furthermore, Ro 25-6981 potentiated the action of levodopa in both species and attenuated the maximal levodopa response in 6-OHDA-lesioned rats chronically treated with levodopa without reducing the overall response. Ro 25-6981 also potentiated the action of the dopamine receptor agonists apomorphine, A68930 and quinpirole in 6-OHDA-lesioned rats. The present observations suggest a therapeutic potential of NR2B-selective NMDA receptor antagonists in the management of PD.
Assuntos
Antiparkinsonianos/uso terapêutico , Transtornos Parkinsonianos/tratamento farmacológico , Fenóis/uso terapêutico , Piperidinas/uso terapêutico , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Apomorfina/farmacologia , Callithrix , Modelos Animais de Doenças , Agonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Levodopa/farmacologia , Masculino , Atividade Motora/efeitos dos fármacos , Oxidopamina , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/patologia , Ratos , Ratos WistarRESUMO
N-Methyl-d-aspartate (NMDA) receptors play key roles in both physiological processes, particularly synaptic plasticity, and in neuropathological states such as epilepsy and acute neurodegeneration. R-(R*,S*)-alpha-(4-Hydroxyphenyl)-beta-methyl-4-(phenyl-methyl)-1-piperidine propanol (RO 25-6981), is a high-affinity and selective blocker of NMDA receptors containing the NR2B subunit. Using site-directed mutagenesis, [3H]RO 25-6981 binding, Xenopus oocyte voltage-clamp recordings, and molecular modeling, we have identified several critical residues involved in the RO 25-6981 binding site within the N-terminal LIVBP-like domain of the human NR2B subunit. Two mutations, NR2B(D101A) and NR2B(F176A), resulted in a complete loss of [3H]RO 25-6981 binding and also abolished the high-affinity RO 25-6981-mediated inhibition of NMDA-induced currents. The mutation NR2B(T233A) led to a marked reduction in binding affinity by 13-fold. Mutations F182A, D104A, or K234A had a more moderate influence on the binding affinity (KD values increased by 8-, 7-, and 6-fold, respectively). In a three-dimensional model of the NR2B LIVBP-like domain based on the X-ray crystal structure of the amino-terminal domain of the mGlu1 receptor, the critical residues are located in the central cleft where interaction with RO 25-6981 may stabilize the closed structure of the domain. Our results suggest that the three amino acids Asp-101, Phe-176, and Thr-233 are important molecular determinants for the high-affinity binding of RO 25-6981 to the LIVBP-like domain of human NR2B. A possible binding mode for RO 25-6981 is proposed.