RESUMO
Neuronal polarity is essential for normal brain development and function. However, cell-intrinsic mechanisms that govern the establishment of neuronal polarity remain to be identified. Here, we report that knockdown of endogenous FOXO proteins in hippocampal and cerebellar granule neurons, including in the rat cerebellar cortex in vivo, reveals a requirement for the FOXO transcription factors in the establishment of neuronal polarity. The FOXO transcription factors, including the brain-enriched protein FOXO6, play a critical role in axo-dendritic polarization of undifferentiated neurites, and hence in a switch from unpolarized to polarized neuronal morphology. We also identify the gene encoding the protein kinase Pak1, which acts locally in neuronal processes to induce polarity, as a critical direct target gene of the FOXO transcription factors. Knockdown of endogenous Pak1 phenocopies the effect of FOXO knockdown on neuronal polarity. Importantly, exogenous expression of Pak1 in the background of FOXO knockdown in both primary neurons and postnatal rat pups in vivo restores the polarized morphology of neurons. These findings define the FOXO proteins and Pak1 as components of a cell-intrinsic transcriptional pathway that orchestrates neuronal polarity, thus identifying a novel function for the FOXO transcription factors in a unique aspect of neural development.
Assuntos
Polaridade Celular/fisiologia , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/citologia , Neurônios/fisiologia , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo , Animais , Polaridade Celular/genética , Células Cultivadas , Fatores de Transcrição Forkhead/genética , Técnicas de Silenciamento de Genes , Hipocampo/fisiologia , Neurônios/metabolismo , Interferência de RNA , RatosRESUMO
Axonal growth is fundamental to the establishment of neuronal connectivity in the brain. However, the cell-intrinsic mechanisms that govern axonal morphogenesis remain to be elucidated. The ubiquitin ligase Cdh1-anaphase-promoting complex (Cdh1-APC) suppresses the growth of axons in postmitotic neurons. Here, we report that Cdh1-APC operates in the nucleus to inhibit axonal growth. We also identify the transcriptional corepressor SnoN as a key target of neuronal Cdh1-APC that promotes axonal growth. Cdh1 forms a physical complex with SnoN and stimulates the ubiquitin-dependent proteasomal degradation of SnoN in neurons. Knockdown of SnoN in neurons significantly reduces axonal growth and suppresses Cdh1 RNAi enhancement of axonal growth. In addition, SnoN knockdown in vivo suggests an essential function for SnoN in the development of granule neuron parallel fibers in the cerebellar cortex. These findings define Cdh1-APC and SnoN as components of a cell-intrinsic pathway that orchestrates axonal morphogenesis in a transcription-dependent manner in the mammalian brain.
Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Diferenciação Celular/fisiologia , Cones de Crescimento/metabolismo , Inibidores do Crescimento/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Encéfalo/citologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Córtex Cerebelar/citologia , Córtex Cerebelar/crescimento & desenvolvimento , Córtex Cerebelar/metabolismo , Regulação para Baixo/fisiologia , Cones de Crescimento/ultraestrutura , Inibidores do Crescimento/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Substâncias Macromoleculares/metabolismo , Proteínas do Tecido Nervoso/genética , Técnicas de Cultura de Órgãos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas/genética , Interferência de RNA/fisiologia , Ratos , Ratos Long-Evans , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Complexos Ubiquitina-Proteína Ligase/genéticaRESUMO
Activation of cyclin-dependent kinase 1 (Cdk1) has been linked to cell death of postmitotic neurons in brain development and disease. We found that Cdk1 phosphorylated the transcription factor FOXO1 at Ser249 in vitro and in vivo. The phosphorylation of FOXO1 at Ser249 disrupted FOXO1 binding with 14-3-3 proteins and thereby promoted the nuclear accumulation of FOXO1 and stimulated FOXO1-dependent transcription, leading to cell death in neurons. In proliferating cells, Cdk1 induced FOXO1 Ser249 phosphorylation at the G2/M phase of the cell cycle, resulting in FOXO1-dependent expression of the mitotic regulator Polo-like kinase (Plk). These findings define a conserved signaling link between Cdk1 and FOXO1 that may have a key role in diverse biological processes, including the degeneration of postmitotic neurons.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclo Celular , Fatores de Transcrição Forkhead/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas 14-3-3/metabolismo , Animais , Apoptose , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Células Cultivadas , Proteína Forkhead Box O1 , Humanos , Camundongos , Células NIH 3T3 , Neurônios/citologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Serina/metabolismo , Transdução de Sinais , Transcrição Gênica , Quinase 1 Polo-LikeRESUMO
Oxidative stress influences cell survival and homeostasis, but the mechanisms underlying the biological effects of oxidative stress remain to be elucidated. Here, we demonstrate that the protein kinase MST1 mediates oxidative-stress-induced cell death in primary mammalian neurons by directly activating the FOXO transcription factors. MST1 phosphorylates FOXO proteins at a conserved site within the forkhead domain that disrupts their interaction with 14-3-3 proteins, promotes FOXO nuclear translocation, and thereby induces cell death in neurons. We also extend the MST-FOXO signaling link to nematodes. Knockdown of the C. elegans MST1 ortholog CST-1 shortens life span and accelerates tissue aging, while overexpression of cst-1 promotes life span and delays aging. The cst-1-induced life-span extension occurs in a daf-16-dependent manner. The identification of the FOXO transcription factors as major and evolutionarily conserved targets of MST1 suggests that MST kinases play important roles in diverse biological processes including cellular responses to oxidative stress and longevity.