RESUMEN
Flavonoids, a group of dietary polyphenols have been shown to possess cognitive health benefits. Epidemiologic evidence suggests that they could play a role in risk reduction in dementia. Amyloid precursor protein processing and the subsequent generation of amyloid beta (Aß) are central to the pathogenesis of Alzheimer's disease, as soluble, oligomeric Aß is thought to be the toxic species driving disease progression. We undertook an in vitro screen to identify flavonoids with bioactivity at ßγ-mediated amyloid precursor protein processing, which lead to identification of a number of flavonoids bioactive at 100 nM. Because of known bioavailability, we investigated the catechin family further and identified epigallocatechin and (-)-epicatechin as potent (nanomolar) inhibitors of amyloidogenic processing. Supporting this finding, we have shown reduced Aß pathology and Aß levels following short term, a 21-day oral delivery of (-)-epicatechin in 7-month-old TASTPM mice. Further, in vitro mechanistic studies suggest this is likely because of indirect BACE1 inhibition. Taken together, our results suggest that orally delivered (-)-epicatechin may be a potential prophylactic for Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/prevención & control , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Catequina/administración & dosificación , Catequina/farmacología , Administración Oral , Animales , Encéfalo/metabolismo , Catequina/análogos & derivados , Células Cultivadas , Progresión de la Enfermedad , Masculino , Ratones TransgénicosRESUMEN
Approximately 15% of human genetic diseases are estimated to involve dysregulation of alternative pre-mRNA splicing. Antisense molecules designed to alter these and other splicing events typically target continuous linear sequences of the message. Here, we show that a structural feature in a pre-mRNA can be targeted by bipartite antisense molecules designed to hybridize with the discontinuous elements that flank the structure and thereby alter splicing. We targeted a hairpin structure at the boundary between exon 10 and intron 10 of the pre-mRNA of tau. Mutations in this region that are associated with certain forms of frontotemporal dementia, destabilize the hairpin to cause increased inclusion of exon 10. Via electrophoretic mobility shift and RNase protection assays, we demonstrate that bipartite antisense molecules designed to simultaneously interact with the available sequences that immediately flank the tau pre-mRNA hairpin do indeed bind to this structured region. Moreover, these agents inhibit exon 10 splicing and reverse the effect of destabilizing disease-causing mutations, in both in vitro splicing assays and cell culture. This general bipartite antisense strategy could be employed to modulate other splicing events that are regulated by RNA secondary structure.
Asunto(s)
Empalme Alternativo , Oligonucleótidos Antisentido/química , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas tau/genética , Línea Celular Tumoral , Humanos , Conformación de Ácido Nucleico , Oligonucleótidos Antisentido/metabolismo , Oligorribonucleótidos/metabolismo , Precursores del ARN/química , ARN Mensajero/química , Proteínas tau/metabolismoRESUMEN
A series of mitoxantrone (MTX) analogues have been designed, synthesized, and evaluated for binding to and stabilizing a stem-loop structure that serves as a splicing regulatory element in the pre-mRNA of tau, which is involved in Alzheimer's and other neurodegenerative diseases. Several compounds showed significantly improved binding activity relative to the original screening hit mitoxantrone. These findings establish essential structure-activity relationships to further optimize the activity of this promising class of compounds.
Asunto(s)
Antraquinonas/síntesis química , Mitoxantrona/análogos & derivados , Mitoxantrona/síntesis química , Poliaminas/síntesis química , Precursores del ARN/química , ARN Mensajero/química , Proteínas tau/genética , Antraquinonas/química , Unión Competitiva , Ligandos , Mitoxantrona/química , Modelos Moleculares , Poliaminas/química , Empalme del ARN , Elementos Reguladores de la Transcripción , Relación Estructura-ActividadRESUMEN
The L-glutamate transporter GLT-1 is an abundant central nervous system (CNS) membrane protein of the excitatory amino acid transporter (EAAT) family that controls extracellular L-glutamate levels and is important in limiting excitotoxic neuronal death. Using reverse transcription-polymerase chain reaction, we have determined that four mRNAs encoding GLT-1 exist in mouse brain, with the potential to encode four GLT-1 isoforms that differ in their N and C termini. We expressed all four isoforms (termed MAST-KREK, MPK-KREK, MAST-DIETCI, and MPK-DIETCI according to amino acid sequence) in a range of cell lines and primary astrocytes and show that each isoform can reach the cell surface. In transfected human embryonic kidney (HEK) 293 or COS-7 cells, all four isoforms support high-affinity sodium-dependent L-glutamate uptake with identical pharmacological and kinetic properties. Inserting a viral epitope (tagged with V5, hemagglutinin, or FLAG) into the second extracellular domain of each isoform allowed coimmunoprecipitation and time-resolved Förster resonance energy transfer (tr-FRET) studies using transfected HEK-293 cells. Here we show for the first time that each of the four isoforms is able to combine to form homomeric and heteromeric assemblies, each of which is expressed at the cell surface of primary astrocytes. After activation of protein kinase C by phorbol ester, V5-tagged GLT-1 is rapidly removed from the cell surface of HEK-293 cells and degraded. This study provides direct biochemical evidence for oligomeric assembly of GLT-1 and reports the development of novel tools to provide insight into the trafficking of GLT-1.
Asunto(s)
Transportador 2 de Aminoácidos Excitadores/química , Animales , Células Cultivadas , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/fisiología , Humanos , Ratones , Isoformas de Proteínas , Proteína Quinasa C/fisiología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
RATIONALE: Nicotine produces behavioural effects that are potentially related to its interaction with diverse nicotinic acetylcholine receptor populations. Evidence from gene deletion studies suggests that the interoceptive stimulus properties of nicotine are mediated by heteromeric high-affinity receptors containing alpha4beta2 subunits. Mice lacking beta2 subunits do not discriminate nicotine (Shoaib et al., Neuropharmacology, 42:530-539, 2002), and nicotine does not elicit dopamine release in these animals (Grady et al., J Neurochem, 76:258-268, 2001). The stimulus properties of nicotine can be detected in rats using a two-lever operant drug discrimination paradigm, allowing them to be classified pharmacologically using ligands with selectivity for receptors containing alpha4beta2, alpha3beta4 or alpha7 subunits. MATERIALS AND METHODS: Rats trained to discriminate 0.4 mg/kg nicotine from vehicle were given the nicotinic receptor agonists, cytisine, varenicline, TC2559, ABT-594, A-85380 (all having high affinity but varying selectivity for alpha4beta2-containing receptors), and WO 03/062224 and WO 01/60821A1 (selective for beta4- and alpha7-containing receptors, respectively). In separate studies, WO 03/062224 was used as the training stimulus. RESULTS: Nicotine, TC-2559, A-85380 and ABT-594 showed dose-dependent and complete stimulus substitution, whilst WO 03/062224 and WO 01/60821A1 were completely without effect. Cytisine and varenicline showed partial generalisation, consistent with their partial agonist activity at nicotinic receptors eliciting dopamine release in rat striatal slices. After almost 50 training sessions with WO 03/062224, there was no clear evidence that an alpha3beta4 receptor agonist could sustain a discriminable stimulus. CONCLUSION: Substitution to the nicotine discriminative stimulus required high-affinity and high intrinsic activity at beta2 but not at beta4- or at alpha7-containing nicotinic receptors.