RESUMO
AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate) recep-tors desensitize rapidly and completely in the continued presence of their endogenous ligand glutamate; however, it is not clear what role AMPA receptor desensitization plays in the brain. We generated a knock-in mouse in which a single amino acid residue, which controls desensitization, was mutated in the GluA2 (GluR2) receptor subunit (GluA2(L483Y)). This mutation was homozygous lethal. However, mice carrying a single mutated allele, GluA2(L483Y/wt), survived past birth, but displayed severe and progressive neurological deficits including seizures and, ultimately, increased mortality. The expression of the AMPA receptor subunits GluA1 and GluA2 was decreased, whereas NMDA receptor protein expression was increased in GluA2(L483Y/wt) mice. Despite this, basal synaptic transmission and plasticity in the hippocampus were largely unaffected, suggesting that neurons preferentially target receptors to synapses to normalize synaptic weight. We found no gross neuroanatomical alterations in GluA2(L483Y/wt) mice. Moreover, there was no accumulation of AMPA receptor subunits in intracellular compartments, suggesting that folding and assembly of AMPA receptors are not affected by this mutation. Interestingly, EPSC paired pulse ratios in the CA1 were enhanced without a change in synaptic release probability, demonstrating that postsynaptic receptor properties can contribute to facilitation. The dramatic phenotype observed in this study by the introduction of a single amino acid change demonstrates an essential role in vivo for AMPA receptor desensitization.
Assuntos
Hipocampo/metabolismo , Doenças do Sistema Nervoso/genética , Fenótipo , Receptores de AMPA/genética , Transmissão Sináptica/fisiologia , Análise de Variância , Animais , Primers do DNA/genética , Eletrofisiologia , Técnicas de Introdução de Genes , Hipocampo/patologia , Immunoblotting , Imuno-Histoquímica , Camundongos , Mutação/genética , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
From our NETSseq-derived human brain transcriptomics data, we identified GPR55 as a potential molecular target for the treatment of motor symptoms in patients with Parkinson's disease. From a high-throughput screen, we identified and optimized agonists with nanomolar potency against both human and rat GPR55. We discovered compounds with either strong or limited ß-arrestin signaling and receptor desensitization, indicating biased signaling. A compound that showed minimal GPR55 desensitization demonstrated a reduction in firing frequency of medium spiny neurons cultured from rat striatum but did not reverse motor deficits in a rat hypolocomotion model. Further profiling of several desensitizing and non-desensitizing lead compounds showed that they are selective over related cannabinoid receptors CB1 and CB2 and that unbound brain concentrations well above the respective GPR55 EC50 can be readily achieved following oral administration. The novel brain-penetrant GPR55 agonists disclosed can be used to probe the role of this receptor in the brain.
Assuntos
Agonistas de Receptores de Canabinoides , Transdução de Sinais , Humanos , Ratos , Animais , Receptores de Canabinoides , beta-Arrestinas , Corpo Estriado/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptor CB2 de Canabinoide , Receptor CB1 de CanabinoideRESUMO
Nicotinic acetylcholine receptor (nAChR) α6 subunit RNA expression is relatively restricted to midbrain regions and is located presynaptically on dopaminergic neurons projecting to the striatum. This subunit modulates dopamine neurotransmission and may have therapeutic potential in movement disorders. We aimed to develop potent and selective α6-containing nAChR antagonists to explore modulation of dopamine release and regulation of motor function in vivo. High-throughput screening (HTS) identified novel α6-containing nAChR antagonists and led to the development of CVN417. This molecule blocks α6-containing nAChR activity in recombinant cells and reduces firing frequency of noradrenergic neurons in the rodent locus coeruleus. CVN417 modulated phasic dopaminergic neurotransmission in an impulse-dependent manner. In a rodent model of resting tremor, CVN417 attenuated this behavioral phenotype. These data suggest that selective antagonism of α6-containing nAChR, with molecules such as CVN417, may have therapeutic utility in treating the movement dysfunctions observed in conditions such as Parkinson's disease.
Assuntos
Dopamina , Receptores Nicotínicos , Encéfalo , Membrana Celular , Corpo Estriado , Antagonistas Nicotínicos/farmacologiaRESUMO
B-ephrin-EphB receptor signaling modulates NMDA receptors by inducing tyrosine phosphorylation of NR2 subunits. Ephrins and EphB RTKs are localized to postsynaptic compartments in the CA1, and therefore potentially interact in a non-canonical cis- configuration. However, it is not known whether cis- configured receptor-ligand signaling is utilized by this class of RTKs, and whether this might influence excitatory synapses. We found that ablation of ephrin-B3 results in an enhancement of the NMDA receptor component of synaptic transmission relative to the AMPA receptor component in CA1 synapses. Synaptic AMPA receptor expression is reduced in ephrin-B3 knockout mice, and there is a marked enhancement of tyrosine phosphorylation of the NR2B receptor subunit. In a reduced system co-expression of ephrin-B3 attenuated EphB2-mediated NR2B tyrosine phosphorylation. Moreover, phosphorylation of EphB2 was elevated in the hippocampus of ephrin-B3 knockout mice, suggesting that regulation of EphB2 activity is lost in these mice. Direct activation of EphB RTKs resulted in phosphorylation of NR2B and a potential signaling partner, the non-receptor tyrosine kinase Pyk2. Our data suggests that ephrin-B3 limits EphB RTK-mediated phosphorylation of the NR2B subunit through an inhibitory cis- interaction which is required for the correct function of glutamatergic CA1 synapses.
Assuntos
Efrina-B3/metabolismo , Hipocampo/metabolismo , Receptores de Glutamato/metabolismo , Transdução de Sinais/fisiologia , Sinapses/metabolismo , Animais , Potenciais Pós-Sinápticos Excitadores , Células HEK293 , Humanos , Immunoblotting , Imunoprecipitação , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Receptores de AMPA/metabolismo , Receptores da Família Eph/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica/fisiologia , TransfecçãoRESUMO
Homocysteine (HCY) is a sulphur-containing amino acid, which has been linked to neurodegenerative diseases such as Alzheimer's disease, and is widely reported to enhance vulnerability of neurons to oxidative, excitotoxic and apoptotic injury via perturbed calcium homeostasis, activation of N-methyl-D-aspartate (NMDA) and metabotropic glutamate (mGlu) receptors. The present study was undertaken to investigate the effects of HCY on long-term potentiation (LTP) and synaptic transmission after chronic 4-week systemic exposure to HCY in adult rats, and possible longer-term effects of HCY 4 weeks after exposure had ended. Contrary to expectation, LTP was enhanced, not retarded after chronic HCY exposure relative to controls. Basic synaptic transmission was not affected at this time point. However, after the 4-week wash out period, a decrease in speed of basic synaptic transmission emerged, and LTP was still partially enhanced, particularly for time points >30 min post-tetanus. In summary, we provide first evidence for sustained HCY-induced changes in hippocampal plasticity and a slow-onset disruption in synaptic transmission. These changes may reflect the suggested (excito-)toxicity of HCY and its putative contribution to neurodegenerative disease.
Assuntos
Hipocampo/efeitos dos fármacos , Homocisteína/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Animais , Hipocampo/fisiologia , Potenciação de Longa Duração/fisiologia , Masculino , Plasticidade Neuronal/fisiologia , Ratos , Transmissão Sináptica/fisiologia , Fatores de TempoRESUMO
1. Group I metabotropic glutamate receptors (mGluRs) are thought to be important modulators of neuronal function in the superior colliculus (SC). Here, we investigated the pharmacology and signalling mechanisms underlying group I mGluR-mediated inhibition of neuronal excitability and synaptic transmission in the rat SC slice. 2. The group I agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) potently depressed synaptically evoked excitatory postsynaptic potentials (EPSPs), currents (EPSCs), and action potentials in a dose-dependent manner (IC50: 6.3 microm). This was strongly reduced by the broad-spectrum antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG, 1 mm, approximately 95% reduction), by the mGluR1 antagonist LY367385 (100 microm, approximately 80% reduction) but not by the mGluR5 antagonist 6-methyl-2-(phenylethynyl)-pyridine (MPEP, 1-100 microm). 3. The putative mGluR5-specific agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, 500 microm) also inhibited EPSPs. Interestingly, CHPG's actions were not blocked by MPEP, but LY367385 (100 microm) reduced the effect of CHPG by 50%. 4. Inhibition induced by DHPG was independent of phospholipase C (PLC)/protein kinase C pathways, and did not require intact intracellular Ca2+ stores. It was not abolished but enhanced by the GABAA antagonist bicuculline (5 microm), suggesting that DHPG's action was not due to facilitated inhibition or changes in neuronal network activity. 5. The K+ channel antagonist 4-aminopyridine (4-AP, 50-100 microm) converted the inhibitory effect of DHPG into facilitation. Paired-pulse depression was strongly reduced by DHPG, an effect that was also prevented by 4-AP. 6. Our data indicate that group I agonists regulate transmitter release, presumably via an autoreceptor in the SC. This receptor may be involved in adaptation to repetitive stimulation via a non-PLC mediated pathway.
Assuntos
Glicina/análogos & derivados , Receptores de Glutamato Metabotrópico/fisiologia , Receptores Pré-Sinápticos/fisiologia , Colículos Superiores/fisiologia , Transmissão Sináptica/fisiologia , 4-Aminopiridina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Relação Dose-Resposta a Droga , Agonistas de Aminoácidos Excitatórios/administração & dosagem , Agonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Glicina/administração & dosagem , Glicina/farmacologia , Técnicas In Vitro , Ativação do Canal Iônico , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Wistar , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Receptores Pré-Sinápticos/agonistas , Receptores Pré-Sinápticos/antagonistas & inibidores , Resorcinóis/administração & dosagem , Resorcinóis/farmacologia , Colículos Superiores/metabolismoRESUMO
Kainate is a potent neurotoxin known to induce acute seizures. However, whether kainate receptors (KARs) play any role in the pathophysiology of temporal lobe epilepsy (TLE) is not known. In TLE, recurrent mossy fiber (rMF) axons form abnormal excitatory synapses onto other dentate granule cells that operate via KARs. The present study explores the pathophysiological implications of KARs in generating recurrent seizures in chronic epilepsy. In an in vitro model of TLE, seizure-like activity was minimized in mice lacking the GluK2 subunit, which is a main component of aberrant synaptic KARs at rMF synapses. In vivo, the frequency of interictal spikes and ictal discharges was strongly reduced in GluK2(-/-) mice or in the presence of a GluK2/GluK5 receptor antagonist. Our data show that aberrant GluK2-containing KARs play a major role in the chronic seizures that characterize TLE and thus constitute a promising antiepileptic target.
Assuntos
Epilepsia do Lobo Temporal/metabolismo , Fibras Musgosas Hipocampais/fisiologia , Receptores de Ácido Caínico/genética , Convulsões/metabolismo , Animais , Epilepsia do Lobo Temporal/fisiopatologia , Potenciais Pós-Sinápticos Excitadores , Masculino , Camundongos , Fibras Musgosas Hipocampais/metabolismo , Receptores de Ácido Caínico/antagonistas & inibidores , Receptores de Ácido Caínico/metabolismo , Convulsões/fisiopatologia , Receptor de GluK2 CainatoRESUMO
Homocysteine (HCY) is a known risk factor for neuronal diseases. We here report that HCY (10-1000 microM) interfered bi-directionally with long-term potentiation (LTP) in hippocampal slices, causing an impairment at concentrations <100 microM, and enhancement > or =500 microM. By comparison, NMDA unidirectionally reduced LTP, whereas l-cysteine led to facilitated LTP. Such HCY-induced alterations in neuronal communication may contribute to cognitive failure in dementia.