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1.
J Pharmacol Exp Ther ; 362(1): 31-44, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28416568

RESUMEN

Alzheimer's disease (AD) is characterized neuropathologically by an abundance of 1) neuritic plaques, which are primarily composed of a fibrillar 42-amino-acid amyloid-ß peptide (Aß), as well as 2) neurofibrillary tangles composed of aggregates of hyperphosporylated tau. Elevations in the concentrations of the Aß42 peptide in the brain, as a result of either increased production or decreased clearance, are postulated to initiate and drive the AD pathologic process. We initially introduced a novel class of bridged aromatics referred tγ-secretase modulatoro as γ-secretase modulators that inhibited the production of the Aß42 peptide and to a lesser degree the Aß40 peptide while concomitantly increasing the production of the carboxyl-truncated Aß38 and Aß37 peptides. These modulators potently lower Aß42 levels without inhibiting the γ-secretase-mediated proteolysis of Notch or causing accumulation of carboxyl-terminal fragments of APP. In this study, we report a large number of pharmacological studies and early assessment of toxicology characterizing a highly potent γ-secretase modulator (GSM), (S)-N-(1-(4-fluorophenyl)ethyl)-6-(6-methoxy-5-(4-methyl-1H-imidazol-1-yl)pyridin-2-yl)-4-methylpyridazin-3-amine (BPN-15606). BPN-15606 displayed the ability to significantly lower Aß42 levels in the central nervous system of rats and mice at doses as low as 5-10 mg/kg, significantly reduce Aß neuritic plaque load in an AD transgenic mouse model, and significantly reduce levels of insoluble Aß42 and pThr181 tau in a three-dimensional human neural cell culture model. Results from repeat-dose toxicity studies in rats and dose escalation/repeat-dose toxicity studies in nonhuman primates have designated this GSM for 28-day Investigational New Drug-enabling good laboratory practice studies and positioned it as a candidate for human clinical trials.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/toxicidad , Fragmentos de Péptidos/antagonistas & inhibidores , Fenetilaminas/farmacología , Fenetilaminas/toxicidad , Piridazinas/farmacología , Piridazinas/toxicidad , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Células Cultivadas , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacocinética , Humanos , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Placa Amiloide/tratamiento farmacológico , Ratas , Ratas Sprague-Dawley , Proteínas tau/metabolismo
2.
Nat Protoc ; 10(7): 985-1006, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-26068894

RESUMEN

Stem cell technologies have facilitated the development of human cellular disease models that can be used to study pathogenesis and test therapeutic candidates. These models hold promise for complex neurological diseases such as Alzheimer's disease (AD), because existing animal models have been unable to fully recapitulate all aspects of pathology. We recently reported the characterization of a novel 3D culture system that exhibits key events in AD pathogenesis, including extracellular aggregation of amyloid-ß (Aß) and accumulation of hyperphosphorylated tau. Here we provide instructions for the generation and analysis of 3D human neural cell cultures, including the production of genetically modified human neural progenitor cells (hNPCs) with familial AD mutations, the differentiation of the hNPCs in a 3D matrix and the analysis of AD pathogenesis. The 3D culture generation takes 1-2 d. The aggregation of Aß is observed after 6 weeks of differentiation, followed by robust tau pathology after 10-14 weeks.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Modelos Neurológicos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Línea Celular , Humanos , Mutación , Agregación Patológica de Proteínas/metabolismo , Proteínas tau/metabolismo
3.
Nature ; 515(7526): 274-8, 2014 Nov 13.
Artículo en Inglés | MEDLINE | ID: mdl-25307057

RESUMEN

Alzheimer's disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-ß plaques and neurofibrillary tangles. The amyloid hypothesis of Alzheimer's disease posits that the excessive accumulation of amyloid-ß peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. Mouse models with familial Alzheimer's disease (FAD) mutations exhibit amyloid-ß-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer's disease, including distinct neurofibrillary tangle pathology. Human neurons derived from Alzheimer's disease patients have shown elevated levels of toxic amyloid-ß species and phosphorylated tau but did not demonstrate amyloid-ß plaques or neurofibrillary tangles. Here we report that FAD mutations in ß-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-ß, including amyloid-ß plaques, in a human neural stem-cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy. Inhibition of amyloid-ß generation with ß- or γ-secretase inhibitors not only decreased amyloid-ß pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-ß-mediated tau phosphorylation. We have successfully recapitulated amyloid-ß and tau pathology in a single 3D human neural cell culture system. Our unique strategy for recapitulating Alzheimer's disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models of other neurodegenerative disorders.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Técnicas de Cultivo de Célula/métodos , Modelos Biológicos , Células-Madre Neurales/metabolismo , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Diferenciación Celular , Evaluación Preclínica de Medicamentos/métodos , Espacio Extracelular/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Células-Madre Neurales/patología , Neuritas/metabolismo , Fosforilación , Presenilina-1/metabolismo , Agregación Patológica de Proteínas , Reproducibilidad de los Resultados , Proteínas tau/química , Proteínas tau/metabolismo
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