RESUMEN
BACKGROUND: Neurofibrillary tangles (NFT), a cardinal neuropathological feature of Alzheimer's disease (AD) that is highly correlated with synaptic loss and dementia severity, appear to be partly attributable to increased phosphorylation of the microtubule stabilizing protein tau at certain AD-related residues. Identifying the kinases involved in the pathologic phosphorylation of tau may provide targets at which to aim new AD-modifying treatments. RESULTS: We report results from a screen of 572 kinases in the human genome for effects on tau hyperphosphorylation using a loss of function, high-throughput RNAi approach. We confirm effects of three kinases from this screen, the eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2), the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A), and the A-kinase anchor protein 13 (AKAP13) on tau phosphorylation at the 12E8 epitope (serine 262/serine 356). We provide evidence that EIF2AK2 effects may result from effects on tau protein expression, whereas DYRK1A and AKAP13 are likely more specifically involved in tau phosphorylation pathways. CONCLUSIONS: These findings identify novel kinases that phosphorylate tau protein and provide a valuable reference data set describing the kinases involved in phosphorylating tau at an AD-relevant epitope.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Proteínas Quinasas/análisis , ARN Interferente Pequeño/análisis , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Línea Celular Tumoral , Perfilación de la Expresión Génica , Pruebas Genéticas , Genoma Humano , Humanos , Fosforilación , Proteínas Quinasas/genética , ARN Interferente Pequeño/genética , Regulación hacia ArribaRESUMEN
High-content analysis methods provide the opportunity to interrogate specific cellular end points in living cells. When coupled with high-throughput RNA interference (ht-RNAi) loss of function screens, high-content analyses are a powerful discovery tool for the identification of new genes and pathways involved in specific disease-relevant cellular functions. The most common readout is a fluorescence measurement, usually based on a green fluorescent protein reporter (or some derivative thereof ) or a fluorescently labeled antibody. Here, we describe a specific approach to the development of a high-content assay for the hyperphosphorylation of tau protein that is compatible with RNAi screens. The goal of this chapter is to provide a generic paradigm, using hyperphosphorylation of tau protein as an example, to serve as a blueprint for the investigation of additional cellular end points or protein functions for those interested in performing high-content screens.
Asunto(s)
Técnica del Anticuerpo Fluorescente , Estudio de Asociación del Genoma Completo/métodos , Interferencia de ARN , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/fisiopatología , Línea Celular Tumoral , Humanos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas tau/genéticaRESUMEN
Technology for high-throughout scanning of the human genome and its encoded proteins have rapidly developed to allow systematic analyses of human disease. Application of these technologies is becoming an increasingly effective approach for identifying the biological basis of genetically complex neurological diseases. This review will highlight significant findings resulting from the use of a multitude of genomic and proteomic technologies toward biomarker discovery in neurological disorders. Though substantial discoveries have been made, there is clearly significant promise and potential remaining to be fully realized through increasing use of and further development of -omic technologies.