RESUMEN
Increased production and deposition of amyloid beta-protein (Abeta) are believed to be key pathogenic events in Alzheimer's disease. As such, routes for lowering cerebral Abeta levels represent potential therapeutic targets for Alzheimer's disease. X11beta is a neuronal adaptor protein that binds to the intracellular domain of the amyloid precursor protein (APP). Overexpression of X11beta inhibits Abeta production in a number of experimental systems. However, whether these changes to APP processing and Abeta production induced by X11beta overexpression also induce beneficial effects to memory and synaptic plasticity are not known. We report here that X11beta-mediated reduction in cerebral Abeta is associated with normalization of both cognition and in vivo long-term potentiation in aged APPswe Tg2576 transgenic mice that model the amyloid pathology of Alzheimer's disease. Overexpression of X11beta itself has no detectable adverse effects upon mouse behaviour. These findings support the notion that modulation of X11beta function represents a therapeutic target for Abeta-mediated neuronal dysfunction in Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Potenciación a Largo Plazo , Memoria , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Conducta Animal , Cadherinas , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Nematode glutamate-gated chloride channels are targets of the macrocyclic lactones, the most important group of anthelmintics available. In Xenopus laevis oocytes, channels formed by the GluClalpha3B subunit from the parasite Haemonchus contortus were more sensitive to l-glutamate (EC(50) = 27.6 +/- 2.7 microM) than those formed by the homologous subunit from Caenorhabditis elegans (EC(50) = 2.2 +/- 0.12 mM). Ibotenate was a partial agonist (EC(50) = 87.7 +/- 3.5 microM). The H. contortus channels responded to low concentrations of ivermectin (estimated EC(50) = approximately 0.1 +/- 1.0 nM), opening slowly and irreversibly in a highly cooperative manner: the rate of channel opening was concentration-dependent. Responses to glutamate and ivermectin were inhibited by picrotoxinin and fipronil. Mutating an N-terminal domain amino acid, leucine 256, to phenylalanine increased the EC(50) for l-glutamate to 92.2 +/- 3.5 microM, and reduced the Hill number from 1.89 +/- 0.35 to 1.09 +/- 0.16. It increased the K(d) for radiolabeled ivermectin binding from 0.35 +/- 0.1 to 2.26 +/- 0.78 nM. Two other mutations (E114G and V235A) had no effect on l-glutamate activation or ivermectin binding: one (T300S) produced no detectable channel activity, but ivermectin binding was similar to wild-type. The substitution of any aromatic amino acid for Leu256 had similar effects in the radioligand binding assay. Molecular modeling studies suggested that the GluCl subunits have a fold similar to that of other Cys-loop ligand-gated ion channels and that amino acid 256 was unlikely to play a direct role in ligand binding but may be involved in mediating the allosteric properties of the receptor.
Asunto(s)
Antihelmínticos/farmacología , Agonistas de los Canales de Cloruro , Haemonchus/metabolismo , Ivermectina/farmacología , Animales , Células COS , Canales de Cloruro/genética , Chlorocebus aethiops , Resistencia a Medicamentos , Femenino , Ácido Glutámico/farmacología , Activación del Canal Iónico , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Oocitos/efectos de los fármacos , Oocitos/fisiología , Técnicas de Placa-Clamp , Conformación Proteica , Subunidades de Proteína/fisiología , Ensayo de Unión Radioligante , Xenopus laevisRESUMEN
Riluzole is the only drug approved for the treatment of amyotrophic lateral sclerosis (ALS) but its precise mode of action is not properly understood. Damage to axonal transport of neurofilaments is believed to be part of the pathogenic mechanism in ALS and this has been linked to defective glutamate handling and increased phosphorylation of neurofilament side-arm domains. Here, we show that riluzole protects against glutamate-induced slowing of neurofilament transport. Protection is associated with decreased neurofilament side-arm phosphorylation and inhibition of the activities of two neurofilament kinases, ERK and p38 that are activated in ALS. Thus, the anti-glutamatergic properties of riluzole include protection against glutamate-induced changes to neurofilament phosphorylation and transport.
Asunto(s)
Transporte Axonal/efectos de los fármacos , Axones/efectos de los fármacos , Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/toxicidad , Proteínas de Neurofilamentos/metabolismo , Fármacos Neuroprotectores/farmacología , Riluzol/farmacología , Análisis de Varianza , Animales , Axones/fisiología , Encéfalo/efectos de los fármacos , Encéfalo/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Inmunohistoquímica , Neuronas/efectos de los fármacos , Neuronas/fisiología , Fosforilación/efectos de los fármacos , Ratas , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Dirofilaria immitis is a filarial nematode that infects dogs and causes cardiopulmonary disease. The most effective way of controlling the infection is by chemoprophylaxis, using members of the avermectin/milbemycin (A/M) class of anthelmintics, which includes ivermectin; these drugs act at invertebrate glutamate-gated chloride channels (GluCl). We have cloned two cDNAs encoding D. immitis GluCl subunits and demonstrated that at least one may be an important molecular target for the A/Ms in vivo. The subunits are orthologues of the alternatively spliced GluClalpha3A and alpha3B subunits (encoded by the avr-14 gene) previously identified in Caenorhabditis elegans and in Haemonchus contortus. Although the alternative splicing of avr-14 is conserved across the species, the processing of the mature GluClalpha3A mRNA differs in D. immitis compared to C. elegans and H. contortus. Two-electrode voltage clamp recordings were made from Xenopus oocytes injected with subunit-specific cRNAs. The DiGluClalpha3B subunit formed channels that were gated by L-glutamate (1-100 mM) and ivermectin (1 microM). Oocytes injected with DiGluClalpha3A cRNA failed to respond to L-glutamate. The qualitative responses obtained were consistent with the pharmacology observed for the GluClalpha3 subunits from C. elegans and H. contortus.
Asunto(s)
Canales de Cloruro/efectos de los fármacos , Dirofilaria immitis/efectos de los fármacos , Filaricidas/farmacología , Ivermectina/farmacología , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Canales de Cloruro/metabolismo , Dirofilaria immitis/genética , Datos de Secuencia Molecular , Oocitos/metabolismo , Técnicas de Placa-Clamp , Alineación de Secuencia , Xenopus/metabolismoRESUMEN
Most of the recent evidence suggests that the avermectin/milbemycin family of anthelmintics act via specific interactions with glutamate-gated chloride channels. These channels are encoded by a small family of genes in nematodes, though the composition of the gene family and the function of the individual members of the family may vary between species. We review our current knowledge concerning the properties of the glutamate-gated chloride channels from Caenorhabditis elegans and the related parasite, Haemonchus contortus. We conclude that the biological effects of the avermectins/milbemycins can be largely explained by the known pharmacology and distribution of the glutamate-gated chloride channels and that differences between the glutamate-gated chloride channels from different nematodes may underlie species-specific variations in anthelmintic action.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Canales de Cloruro/metabolismo , Haemonchus/fisiología , Ivermectina/análogos & derivados , Animales , Antihelmínticos/uso terapéutico , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/farmacología , Canales de Cloruro/farmacología , Haemonchus/efectos de los fármacos , Humanos , Ivermectina/uso terapéutico , Movimiento/fisiología , Infecciones por Nematodos/tratamiento farmacológico , Faringe/metabolismoRESUMEN
X11alpha is a neuronal-specific adaptor protein that binds to the amyloid-beta protein precursor (AbetaPP). Overexpression of X11alpha reduces Abeta production but whether X11alpha also protects against Abeta-related memory dysfunction is not known. To test this possibility, we crossed X11alpha transgenic mice with AbetaPP-Tg2576 mice. AbetaPP-Tg2576 mice produce high levels of brain Abeta and develop age-related defects in memory function that correlate with increasing Abeta load. Overexpression of X11alpha alone had no detectable adverse effect upon behavior. However, X11alpha reduced brain Abeta levels and corrected spatial reference memory defects in aged X11alpha/AbetaPP double transgenics. Thus, X11alpha may be a therapeutic target for Alzheimer's disease.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Enfermedad de Alzheimer/complicaciones , Trastornos de la Memoria , Proteínas del Tejido Nervioso/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Factores de Edad , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Humanos , Aprendizaje por Laberinto/fisiología , Trastornos de la Memoria/etiología , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/prevención & control , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genéticaRESUMEN
Neurofilaments are the intermediate filaments of neurons and are synthesised in neuronal cell bodies and then transported through axons. Neurofilament light chain (NFL) is a principal component of neurofilaments, and phosphorylation of NFL head domain is believed to regulate the assembly of neurofilaments. However, the role that NFL phosphorylation has on transport of neurofilaments is poorly understood. To address this issue, we monitored axonal transport of phosphorylation mutants of NFL. We mutated four known phosphorylation sites in NFL head domain to either preclude phosphorylation, or mimic permanent phosphorylation. Mutation to preclude phosphorylation had no effect on transport but mutation of three sites to mimic permanent phosphorylation inhibited transport. Mutation of all four sites together to mimic permanent phosphorylation proved especially potent at inhibiting transport and also disrupted neurofilament assembly. Our results suggest that NFL head domain phosphorylation is a regulator of neurofilament axonal transport.
Asunto(s)
Transporte Axonal , Axones/metabolismo , Filamentos Intermedios/metabolismo , Neuronas/metabolismo , Animales , Células Cultivadas , Filamentos Intermedios/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neuronas/citología , Fosforilación , Ratas , TransfecciónRESUMEN
We have isolated a cDNA from Dirofilaria immitis that encodes a predicted ion channel subunit, Di-LGR-1. Secondary structure predictions and database searches reveal that Di-LGR-1 is distantly related to ligand-gated anion channels, such as the GABA(A) receptors, though there are marked differences in the sequences of the putative channel forming regions. Di-LGR-1 has 52% sequence identity to the Caenorhabditis elegans predicted polypeptide, T27A1.4: neighbour-joining trees show that these two polypeptides are the most divergent members of the nematode ligand-gated anion channel family. No close homologues are present in vertebrates, suggesting that their function may be specific to nematodes. RNAi experiments using a fragment of T27A1.4 with C. elegans failed to reveal any obvious phenotype, so the function of these channels remains unknown.