RESUMEN
Aggregates of hyperphosphorylated tau protein are a pathological hallmark of more than 20 distinct neurodegenerative diseases, including Alzheimer's disease, progressive supranuclear palsy, and frontotemporal dementia. While the exact mechanism of tau aggregation is unknown, the accumulation of aggregates correlates with disease progression. Here we report a genome-wide CRISPR screen to identify modulators of endogenous tau protein for the first time. Primary screens performed in SH-SY5Y cells, identified positive and negative regulators of tau protein levels. Hit validation of the top 43 candidate genes was performed using Ngn2-induced human cortical excitatory neurons. Using this approach, genes and pathways involved in modulation of endogenous tau levels were identified, including chromatin modifying enzymes, neddylation and ubiquitin pathway members, and components of the mTOR pathway. TSC1, a critical component of the mTOR pathway, was further validated in vivo, demonstrating the relevance of this screening strategy. These findings may have implications for treating neurodegenerative diseases in the future.
Asunto(s)
Redes y Vías Metabólicas/genética , Neuronas/metabolismo , Proteínas tau/metabolismo , Animales , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Línea Celular Tumoral , Edición Génica , Genes/genética , Genes/fisiología , Pruebas Genéticas/métodos , Estudio de Asociación del Genoma Completo , Humanos , Ratones , Neuroblastoma/metabolismo , Ratas , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Complex regulatory networks regulate stem cell behavior and contributions to tissue growth, repair, and homeostasis. A full understanding of the networks controlling stem cell self-renewal and differentiation, however, has not yet been realized. To systematically dissect these networks and identify their components, we performed an unbiased, transcriptome-wide in vivo RNAi screen in female Drosophila germline stem cells (GSCs). Based on characterized cellular defects, we classified 646 identified genes into phenotypic and functional groups and unveiled a comprehensive set of networks regulating GSC maintenance, survival, and differentiation. This analysis revealed an unexpected role for ribosomal assembly factors in controlling stem cell cytokinesis. Moreover, our data show that the transition from self-renewal to differentiation relies on enhanced ribosome biogenesis accompanied by increased protein synthesis. Collectively, these results detail the extensive genetic networks that control stem cell homeostasis and highlight the intricate regulation of protein synthesis during differentiation.
Asunto(s)
Diferenciación Celular , Drosophila melanogaster/citología , Células Germinativas/citología , Biogénesis de Organelos , Biosíntesis de Proteínas , Ribosomas/metabolismo , Células Madre/citología , Animales , Nucléolo Celular/patología , Supervivencia Celular/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Factor 4E Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genes de Insecto , Hipertrofia , Iniciación de la Cadena Peptídica Traduccional/genética , Fenotipo , Unión Proteica , Interferencia de ARN , Transcriptoma/genéticaRESUMEN
The Drosophila melanogaster ovary is a powerful, genetically tractable system through which one can elucidate the principles underlying cellular function and organogenesis in vivo. In order to understand the intricate process of oogenesis at the subcellular level, microscopic analysis with the highest possible resolution is required. In this chapter, we describe the preparation of ovaries for ultrastructural analysis using transmission electron microscopy and focused ion beam scanning electron microscopy. We discuss and provide protocols for chemical fixation of Drosophila ovaries that facilitate optimal imaging with particular attention paid to preserving and resolving mitochondrial membrane morphology and structure.
Asunto(s)
Microscopía Electrónica de Transmisión/métodos , Oogénesis , Ovario/ultraestructura , Animales , Drosophila melanogaster , FemeninoRESUMEN
The differentiation of stem cells is a tightly regulated process essential for animal development and tissue homeostasis. Through this process, attainment of new identity and function is achieved by marked changes in cellular properties. Intrinsic cellular mechanisms governing stem cell differentiation remain largely unknown, in part because systematic forward genetic approaches to the problem have not been widely used. Analysing genes required for germline stem cell differentiation in the Drosophila ovary, we find that the mitochondrial ATP synthase plays a critical role in this process. Unexpectedly, the ATP synthesizing function of this complex was not necessary for differentiation, as knockdown of other members of the oxidative phosphorylation system did not disrupt the process. Instead, the ATP synthase acted to promote the maturation of mitochondrial cristae during differentiation through dimerization and specific upregulation of the ATP synthase complex. Taken together, our results suggest that ATP synthase-dependent crista maturation is a key developmental process required for differentiation independent of oxidative phosphorylation.
