RESUMEN
Mutations in MAPT (microtubule-associated protein tau) cause frontotemporal dementia (FTD). MAPT mutations are associated with abnormal tau phosphorylation levels and accumulation of misfolded tau protein that can propagate between neurons ultimately leading to cell death (tauopathy). Recently, a p.A152T tau variant was identified as a risk factor for FTD, Alzheimer's disease, and synucleinopathies. Here we used induced pluripotent stem cells (iPSC) from a patient carrying this p.A152T variant to create a robust, functional cellular assay system for probing pathophysiological tau accumulation and phosphorylation. Using stably transduced iPSC-derived neural progenitor cells engineered to enable inducible expression of the pro-neural transcription factor Neurogenin 2 (Ngn2), we generated disease-relevant, cortical-like glutamatergic neurons in a scalable, high-throughput screening compatible format. Utilizing automated confocal microscopy, and an advanced image-processing pipeline optimized for analysis of morphologically complex human neuronal cultures, we report quantitative, subcellular localization-specific effects of multiple kinase inhibitors on tau, including ones under clinical investigation not previously reported to affect tau phosphorylation. These results demonstrate the potential for using patient iPSC-derived ex vivo models of tauopathy as genetically accurate, disease-relevant systems to probe tau biochemistry and support the discovery of novel therapeutics for tauopathies.
Asunto(s)
Glutamatos/metabolismo , Procesamiento de Imagen Asistido por Computador , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Neuronas/patología , Proteómica , Tauopatías/patología , Proteínas tau/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biomarcadores/metabolismo , Línea Celular , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteínas Quinasas/metabolismo , Piridinas/química , Piridinas/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Using structure-guided design, several cell based assays, and microdosed positron emission tomography (PET) imaging, we identified a series of highly potent, selective, and brain-penetrant oxazole-4-carboxamide-based inhibitors of glycogen synthase kinase-3 (GSK-3). An isotopologue of our first-generation lead, [3H]PF-367, demonstrates selective and specific target engagement in vitro, irrespective of the activation state. We discovered substantial ubiquitous GSK-3-specific radioligand binding in Tg2576 Alzheimer's disease (AD), suggesting application for these compounds in AD diagnosis and identified [11C]OCM-44 as our lead GSK-3 radiotracer, with optimized brain uptake by PET imaging in nonhuman primates. GSK-3ß-isozyme selectivity was assessed to reveal OCM-51, the most potent (IC50 = 0.030 nM) and selective (>10-fold GSK-3ß/GSK-3α) GSK-3ß inhibitor known to date. Inhibition of CRMP2T514 and tau phosphorylation, as well as favorable therapeutic window against WNT/ß-catenin signaling activation, was observed in cells.
Asunto(s)
Encéfalo/metabolismo , Descubrimiento de Drogas , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Tomografía de Emisión de Positrones/métodos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/diagnóstico por imagen , Dominio Catalítico , Glucógeno Sintasa Quinasa 3 beta/química , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Neuroimagen , Oxazoles/química , Oxazoles/metabolismo , Oxazoles/farmacología , Inhibidores de Proteínas Quinasas/metabolismo , Triazoles/química , Triazoles/metabolismo , Triazoles/farmacologíaRESUMEN
Histone deacetylases (HDACs) are enzymes involved in the epigenetic control of gene expression. A handful of HDAC inhibitors have been approved for the treatment of cancer, and HDAC inhibition has also been proposed as a novel therapeutic strategy for neurodegenerative disorders. These disorders include progranulin (PGRN)-deficient forms of frontotemporal dementia caused by mutations in the GRN gene that lead to haploinsufficiency. Hydroxamic-acid-based inhibitors of HDACs 1-3, reported to have fast-on/fast-off binding kinetics, induce increased expression of PGRN in human neuronal models, while the benzamide class of slow-binding HDAC inhibitors does not produce this effect. These observations indicate that the kinetics of HDAC inhibitor binding can be tuned for optimal induction of human PGRN expression in neurons. Here, we further expand on these findings using human cortical-like, glutamatergic neurons. We provide evidence that two prototypical, potent hydroxamic acid HDAC inhibitors that induce PGRN (panobinostat and trichostatin A) exhibit an initial fast-binding step followed by a second, slower step, referred to as mechanism B of slow binding, rather than simpler fast-on/fast-off binding kinetics. In addition, we show that trapoxin A, a macrocyclic, epoxyketone-containing class I HDAC inhibitor, exhibits slow binding with high, picomolar potency and also induces PGRN expression in human neurons. Finally, we demonstrate induction of PGRN expression by fast-on/fast-off, highly potent, macrocyclic HDAC inhibitors with ethyl ketone or ethyl ester Zn2+ binding groups. Taken together, these data expand our understanding of HDAC1-3 inhibitor binding kinetics, and further delineate the specific combinations of structural and kinetic features of HDAC inhibitors that are optimal for upregulating PGRN expression in human neurons and thus may have translational relevance in neurodegenerative disease.
Asunto(s)
Inhibidores de Histona Desacetilasas/farmacocinética , Ácidos Hidroxámicos/farmacocinética , Neuronas/efectos de los fármacos , Panobinostat/farmacocinética , Progranulinas/metabolismo , Demencia Frontotemporal/metabolismo , Expresión Génica/efectos de los fármacos , Ácido Glutámico/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Células-Madre Neurales , Neuronas/metabolismo , Péptidos/farmacocinéticaRESUMEN
Identifying the mechanisms through which genetic risk causes dementia is an imperative for new therapeutic development. Here, we apply a multistage, systems biology approach to elucidate the disease mechanisms in frontotemporal dementia. We identify two gene coexpression modules that are preserved in mice harboring mutations in MAPT, GRN and other dementia mutations on diverse genetic backgrounds. We bridge the species divide via integration with proteomic and transcriptomic data from the human brain to identify evolutionarily conserved, disease-relevant networks. We find that overexpression of miR-203, a hub of a putative regulatory microRNA (miRNA) module, recapitulates mRNA coexpression patterns associated with disease state and induces neuronal cell death, establishing this miRNA as a regulator of neurodegeneration. Using a database of drug-mediated gene expression changes, we identify small molecules that can normalize the disease-associated modules and validate this experimentally. Our results highlight the utility of an integrative, cross-species network approach to drug discovery.
Asunto(s)
Demencia/genética , Evolución Molecular , Redes Reguladoras de Genes , Enfermedades Neurodegenerativas/genética , Animales , Muerte Celular/genética , Modelos Animales de Enfermedad , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Vectores Genéticos/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/genética , MicroARNs/metabolismo , Proteómica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Transcriptoma/genética , Proteínas tau/metabolismoRESUMEN
The ubiquitin-proteasome system (UPS) is responsible for most selective protein degradation in eukaryotes and regulates numerous cellular processes, including cell cycle control and protein quality control. A component of this system, the deubiquitinating enzyme USP14, associates with the proteasome where it can rescue substrates from degradation by removal of the ubiquitin tag. We previously found that a small-molecule inhibitor of USP14, known as IU1, can increase the rate of degradation of a subset of proteasome substrates. We report here the synthesis and characterization of 87 variants of IU1, which resulted in the identification of a 10-fold more potent USP14 inhibitor that retains specificity for USP14. The capacity of this compound, IU1-47, to enhance protein degradation in cells was tested using as a reporter the microtubule-associated protein tau, which has been implicated in many neurodegenerative diseases. Using primary neuronal cultures, IU1-47 was found to accelerate the rate of degradation of wild-type tau, the pathological tau mutants P301L and P301S, and the A152T tau variant. We also report that a specific residue in tau, lysine 174, is critical for the IU1-47-mediated tau degradation by the proteasome. Finally, we show that IU1-47 stimulates autophagic flux in primary neurons. In summary, these findings provide a powerful research tool for investigating the complex biology of USP14.
Asunto(s)
Embrión de Mamíferos/metabolismo , Inhibidores Enzimáticos/farmacología , Fibroblastos/metabolismo , Neuronas/metabolismo , Pirroles/farmacología , Ubiquitina Tiolesterasa/fisiología , Proteínas tau/metabolismo , Animales , Células Cultivadas , Citoplasma/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/citología , Neuronas/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Pirroles/síntesis química , Ratas Sprague-Dawley , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Reprogramming of human somatic cells into induced pluripotent stem (iPS) cells has greatly expanded the set of research tools available to investigate the molecular and cellular mechanisms underlying central nervous system (CNS) disorders. Realizing the promise of iPS cell technology for the identification of novel therapeutic targets and for high-throughput drug screening requires implementation of methods for the large-scale production of defined CNS cell types. Here we describe a protocol for generating stable, highly expandable, iPS cell-derived CNS neural progenitor cells (NPC) using multi-dimensional fluorescence activated cell sorting (FACS) to purify NPC defined by cell surface markers. In addition, we describe a rapid, efficient, and reproducible method for generating excitatory cortical-like neurons from these NPC through inducible expression of the pro-neural transcription factor Neurogenin 2 (iNgn2-NPC). Finally, we describe methodology for the use of iNgn2-NPC for probing human neuroplasticity and mechanisms underlying CNS disorders using high-content, single-cell-level automated microscopy assays. © 2017 by John Wiley & Sons, Inc.
Asunto(s)
Diferenciación Celular , Enfermedades del Sistema Nervioso Central/tratamiento farmacológico , Enfermedades del Sistema Nervioso Central/patología , Ensayos Analíticos de Alto Rendimiento , Células Madre Pluripotentes Inducidas/citología , Modelos Biológicos , Células-Madre Neurales/citología , Neuronas/citología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular , Proliferación Celular , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/metabolismo , Plasticidad Neuronal , Neuronas/metabolismo , Análisis de la Célula IndividualRESUMEN
Frontotemporal dementia (FTD) and other tauopathies characterized by focal brain neurodegeneration and pathological accumulation of proteins are commonly associated with tau mutations. However, the mechanism of neuronal loss is not fully understood. To identify molecular events associated with tauopathy, we studied induced pluripotent stem cell (iPSC)-derived neurons from individuals carrying the tau-A152T variant. We highlight the potential of in-depth phenotyping of human neuronal cell models for pre-clinical studies and identification of modulators of endogenous tau toxicity. Through a panel of biochemical and cellular assays, A152T neurons showed accumulation, redistribution, and decreased solubility of tau. Upregulation of tau was coupled to enhanced stress-inducible markers and cell vulnerability to proteotoxic, excitotoxic, and mitochondrial stressors, which was rescued upon CRISPR/Cas9-mediated targeting of tau or by pharmacological activation of autophagy. Our findings unmask tau-mediated perturbations of specific pathways associated with neuronal vulnerability, revealing potential early disease biomarkers and therapeutic targets for FTD and other tauopathies.
Asunto(s)
Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Neuronas/metabolismo , Proteínas tau/genética , Sustitución de Aminoácidos , Autofagia/genética , Biomarcadores , Diferenciación Celular , Línea Celular , Codón , Demencia Frontotemporal/patología , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neuronas/citología , Isoformas de Proteínas , Procesamiento Proteico-Postraduccional , Estrés Fisiológico , Proteínas tau/metabolismoRESUMEN
Wnt/ß-catenin signaling has emerged as a central player in pathways implicated in the pathophysiology and treatment of neuropsychiatric disorders. To identify potential novel therapeutics for these disorders, high-throughput screening (HTS) assays reporting on Wnt/ß-catenin signaling in disease-relevant contexts are needed. The use of human patient-derived induced pluripotent stem cell (iPSC) models provides ideal disease-relevant context if these stem cell cultures can be adapted for HTS-compatible formats. Here, we describe a sensitive, HTS-compatible Wnt/ß-catenin signaling reporter system generated in homogeneous, expandable neural progenitor cells (NPCs) derived from human iPSCs. We validated this system by demonstrating dose-responsive stimulation by several known Wnt/ß-catenin signaling pathway modulators, including Wnt3a, a glycogen synthase kinase-3 (GSK3) inhibitor, and the bipolar disorder therapeutic lithium. These responses were robust and reproducible over time across many repeated assays. We then conducted a screen of ~1500 compounds from a library of Food and Drug Administration-approved drugs and known bioactives and confirmed the HTS hits, revealing multiple chemical and biological classes of novel small-molecule probes of Wnt/ß-catenin signaling. Generating these type of pathway-selective, cell-based phenotypic assays in human iPSC-derived neural cells will advance the field of human experimental neurobiology toward the goal of identifying and validating targets for neuropsychiatric disorders.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Células-Madre Neurales/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Litio/farmacología , Trastornos Mentales/tratamiento farmacológico , Trastornos Mentales/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/farmacología , Proteína Wnt3A/metabolismo , beta Catenina/metabolismoRESUMEN
The objective of this study was to elucidate the role of fat distribution in predicting insulin resistance in peri- or post-menopausal women. The results demonstrated that insulin resistance increases with waist circumference and subscapular skinfold thickness but decreases with higher thigh circumflex in the peri- and post-menopausal women.
Asunto(s)
Constitución Corporal , Resistencia a la Insulina , Posmenopausia , Tejido Adiposo/anatomía & histología , Anciano , Brazo/anatomía & histología , Femenino , Humanos , Persona de Mediana Edad , Grosor de los Pliegues Cutáneos , Muslo/anatomía & histología , Circunferencia de la CinturaRESUMEN
The glutamate transporter excitatory amino acid transporter 3 (EAAT3) is polarized to the apical surface in epithelial cells and localized to the dendritic compartment in hippocampal neurons, where it is clustered adjacent to postsynaptic sites. In this study, we analyzed the sequences in EAAT3 that are responsible for its polarized localization in Madin-Darby canine kidney (MDCK) cells and neurons. Confocal microscopy and cell surface biotinylation assays demonstrated that deletion of the EAAT3 C terminus or replacement of the C terminus of EAAT3 with the analogous region in EAAT1 eliminated apical localization in MDCK cells. The C terminus of EAAT3 was sufficient to redirect the basolateral-preferring EAAT1 and the nonpolarized EAAT2 to the apical surface. Using alanine substitution mutants, we identified a short peptide motif in the cytoplasmic C-terminal region of EAAT3 that directs its apical localization in MDCK cells. Mutation of this sequence also impairs dendritic targeting of EAAT3 in hippocampal neurons but does not interfere with the clustering of EAAT3 on dendritic spines and filopodia. These data provide the first evidence that an identical cytoplasmic motif can direct apical targeting in epithelia and somatodendritic targeting in neurons. Moreover, our results demonstrate that the two fundamental features of the localization of EAAT3 in neurons, its restriction to the somatodendritic domain and its clustering near postsynaptic sites, are mediated by distinct molecular mechanisms.