RESUMO
INTRODUCTION: Tuberous sclerosis complex (TSC) is a genetic disease resulting from mutation in TSC1 or TSC2 and subsequent hyperactivation of mammalian Target of Rapamycin (mTOR). Common TSC features include brain lesions, such as cortical tubers and subependymal giant cell astrocytomas (SEGAs). However, the current treatment with mTOR inhibitors has critical limitations. We aimed to identify new targets for TSC pharmacotherapy. RESULTS: The results of our shRNA screen point to glutamate-cysteine ligase catalytic subunit (GCLC), a key enzyme in glutathione synthesis, as a contributor to TSC-related phenotype. GCLC inhibition increased cellular stress and reduced mTOR hyperactivity in TSC2-depleted neurons and SEGA-derived cells. Moreover, patients' brain tubers showed elevated GCLC and stress markers expression. Finally, GCLC inhibition led to growth arrest and death of SEGA-derived cells. CONCLUSIONS: We describe GCLC as a part of redox adaptation in TSC, needed for overgrowth and survival of mutant cells, and provide a potential novel target for SEGA treatment.
Assuntos
Encéfalo/patologia , Glutamato-Cisteína Ligase/metabolismo , Neurônios/metabolismo , Esclerose Tuberosa/patologia , Adolescente , Animais , Butionina Sulfoximina/farmacologia , Células COS , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Criança , Chlorocebus aethiops , Inibidores Enzimáticos/farmacologia , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunossupressores/farmacologia , Masculino , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Adulto JovemRESUMO
The shape of the dendritic arbor is one of the criteria of neuron classification and reflects functional specialization of particular classes of neurons. The development of a proper dendritic branching pattern strongly relies on interactions between the extracellular environment and intracellular processes responsible for dendrite growth and stability. We previously showed that mammalian target of rapamycin (mTOR) kinase is crucial for this process. In this work, we performed a screen for modifiers of dendritic growth in hippocampal neurons, the expression of which is potentially regulated by mTOR. As a result, we identified Cyr61, an angiogenic factor with unknown neuronal function, as a novel regulator of dendritic growth, which controls dendritic growth in a ß1-integrin-dependent manner.
Assuntos
Proteína Rica em Cisteína 61/fisiologia , Dendritos/fisiologia , Matriz Extracelular/metabolismo , Hipocampo/citologia , Neurônios/fisiologia , Animais , Forma Celular , Células Cultivadas , Proteína Rica em Cisteína 61/genética , Proteína Rica em Cisteína 61/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Precoces , Hipocampo/metabolismo , Insulina/fisiologia , Integrina beta1/metabolismo , Integrina beta1/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , RNA Interferente Pequeno/genética , Ratos , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteínas ras/metabolismo , Proteínas ras/fisiologiaRESUMO
Dendrites are the main site of information input into neurons. Their development is a multistep process controlled by mammalian target of rapamycin (mTOR) among other proteins. mTOR is a serine/threonine protein kinase that forms two functionally distinct complexes in mammalian cells: mTORC1 and mTORC2. However, the one that contributes to mammalian neuron development remains unknown. This work used short hairpin RNA against Raptor and Rictor, unique components of mTORC1 and mTORC2, respectively, to dissect mTORC involvement in this process. We provide evidence that both mTOR complexes are crucial for the proper dendritic arbor morphology of hippocampal neurons. These two complexes are required for dendritic development both under basal conditions and upon the induction of mTOR-dependent dendritic growth. We also identified Akt as a downstream effector of mTORC2 needed for proper dendritic arbor morphology, the action of which required mTORC1 and p70S6K1.
