RESUMEN

B23, also known as nucleophosmin (NPM), is multifunctional protein directly implicated in cell proliferation, cell cycle progression, and cell survival. In the current study, in addition to confirming its anti-apoptotic function in neuronal survival, we demonstrated that the spatial-temporal expression profile of B23 during development of hippocampal neurons is high in the embryonic stage, down-regulated after birth, and preferentially localized at the tips of growing neuritis and branching points. Overexpression of B23 promotes axon growth with abundant branching points in growing hippocampal neurons, but depletion of B23 impairs axon growth, leading to neuronal death. Following injury to the trisynaptic path in hippocampal slice, overexpression of B23 remarkably increased the number and length of regenerative fibers in the mossy fiber path. Our study suggests that B23 expression in developing neurons is essential for neuritogenesis and axon growth and that up-regulation of B23 may be a strategy for enhancing the reconstitution of synaptic paths after injury to hippocampal synapses.

Asunto(s)

Hipocampo/lesiones , Hipocampo/metabolismo , Proteínas Nucleares/metabolismo , Sinapsis/metabolismo , Animales , Axones/metabolismo , Muerte Celular , Ratones , Fibras Musgosas del Hipocampo/metabolismo , Fibras Musgosas del Hipocampo/patología , Regeneración Nerviosa , Nucleofosmina , Ratas

RESUMEN

Mechanistic studies of axon growth during development are beneficial to the search for neuron-intrinsic regulators of axon regeneration. Here, we discovered that, in the developing neuron from rat, Akt signaling regulates axon growth and growth cone formation through phosphorylation of serine 14 (S14) on Inhibitor of DNA binding 2 (Id2). This enhances Id2 protein stability by means of escape from proteasomal degradation, and steers its localization to the growth cone, where Id2 interacts with radixin that is critical for growth cone formation. Knockdown of Id2, or abrogation of Id2 phosphorylation at S14, greatly impairs axon growth and the architecture of growth cone. Intriguingly, reinstatement of Akt/Id2 signaling after injury in mouse hippocampal slices redeemed growth promoting ability, leading to obvious axon regeneration. Our results suggest that Akt/Id2 signaling is a key module for growth cone formation and axon growth, and its augmentation plays a potential role in CNS axonal regeneration.

Asunto(s)

Axones/fisiología , Sistema Nervioso Central/citología , Conos de Crecimiento/fisiología , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Regeneración , Animales , Proteínas del Citoesqueleto/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Fosforilación , Procesamiento Proteico-Postraduccional , Ratas

RESUMEN

Neurofibrillary tangles (NFTs), composed of truncated and hyperphosphorylated tau, are a common feature of numerous aging-related neurodegenerative diseases, including Alzheimer's disease (AD). However, the molecular mechanisms mediating tau truncation and aggregation during aging remain elusive. Here we show that asparagine endopeptidase (AEP), a lysosomal cysteine proteinase, is activated during aging and proteolytically degrades tau, abolishes its microtubule assembly function, induces tau aggregation and triggers neurodegeneration. AEP is upregulated and active during aging and is activated in human AD brain and tau P301S-transgenic mice with synaptic pathology and behavioral impairments, leading to tau truncation in NFTs. Tau P301S-transgenic mice with deletion of the gene encoding AEP show substantially reduced tau hyperphosphorylation, less synapse loss and rescue of impaired hippocampal synaptic function and cognitive deficits. Mice infected with adeno-associated virus encoding an uncleavable tau mutant showed attenuated pathological and behavioral defects compared to mice injected with adeno-associated virus encoding tau P301S. Together, these observations indicate that AEP acts as a crucial mediator of tau-related clinical and neuropathological changes. Inhibition of AEP may be therapeutically useful for treating tau-mediated neurodegenerative diseases.

Asunto(s)

Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/patología , Cisteína Endopeptidasas/metabolismo , Ovillos Neurofibrilares/enzimología , Ovillos Neurofibrilares/patología , Proteínas tau/metabolismo , Anciano , Envejecimiento/patología , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/fisiopatología , Secuencia de Aminoácidos , Animales , Asparagina/metabolismo , Encéfalo/enzimología , Encéfalo/patología , Calpaína/metabolismo , Caspasas/metabolismo , Cognición , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Trastornos de la Memoria/complicaciones , Trastornos de la Memoria/patología , Trastornos de la Memoria/fisiopatología , Ratones Transgénicos , Datos de Secuencia Molecular , Neurotoxinas/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Solubilidad , Sinapsis/metabolismo , Sinapsis/patología , Regulación hacia Arriba , Proteínas tau/química

RESUMEN

AIMS: Vascular endothelial dysfunction and inflammation are hallmarks of atherosclerosis. Krüppel-like factor 2 (KLF2) is a key mediator of anti-inflammatory and anti-atherosclerotic properties of the endothelium. However, little is known of the molecular mechanisms for regulating KLF2 transcriptional activation. METHODS AND RESULTS: Here, we found that histone deacetylase 5 (HDAC5) associates with KLF2 and represses KLF2 transcriptional activation. HDAC5 resided with KLF2 in the nuclei of human umbilical cord vein endothelial cells (HUVECs). Steady laminar flow attenuated the association of HDAC5 with KLF2 via stimulating HDAC5 phosphorylation-dependent nuclear export in HUVEC. We also mapped the KLF2-HDAC5-interacting domains and found that the N-terminal region of HDAC5 interacts with the C-terminal domain of KLF2. Chromatin immunoprecipitation and luciferase reporter assays showed that HDAC5 through a direct association with KLF2 suppressed KLF2 transcriptional activation. HDAC5 overexpression inhibited KLF2-dependent endothelial nitric oxide synthesis (eNOS) promoter activity in COS7 cell and gene expression in both HUVECs and bovine aortic endothelial cells (BAECs). Conversely, HDAC5 silencing enhanced KLF2 transcription and hence eNOS expression in HUVEC. Moreover, we observed that the level of eNOS protein in the thoracic aorta isolated from HDAC5 knockout mice was higher, whereas expression of pro-inflammatory vascular cell adhesion molecule 1 was lower, compared with those of HDAC5 wild-type mice. CONCLUSIONS: We reveal a novel role of HDAC5 in modulating the KLF2 transcriptional activation and eNOS expression. These findings suggest that HDAC5, a binding partner and modulator of KLF2, could be a new therapeutic target to prevent vascular endothelial dysfunction associated with cardiovascular diseases.

Asunto(s)

Histona Desacetilasas/metabolismo , Células Endoteliales de la Vena Umbilical Humana/enzimología , Factores de Transcripción de Tipo Kruppel/metabolismo , Transcripción Genética , Transporte Activo de Núcleo Celular , Animales , Células COS , Bovinos , Chlorocebus aethiops , Regulación Enzimológica de la Expresión Génica , Histona Desacetilasas/deficiencia , Histona Desacetilasas/genética , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estrés Mecánico , Activación Transcripcional , Transfección

RESUMEN

Serine-arginine protein kinases 2 (SRPK2) is a cell cycle-regulated kinase that phosphorylates serine/arginine domain-containing proteins and mediates pre-mRNA splicing with unclear function in neurons. Here, we show that SRPK2 phosphorylates tau on S214, suppresses tau-dependent microtubule polymerization, and inhibits axonal elongation in neurons. Depletion of SRPK2 in dentate gyrus inhibits tau phosphorylation in APP/PS1 mouse and alleviates the impaired cognitive behaviors. The defective LTP in APP/PS1 mice is also improved after SRPK2 depletion. Moreover, active SRPK2 is increased in the cortex of APP/PS1 mice and the pathological structures of human Alzheimer's disease (AD) brain. Therefore, our study suggests SRPK2 may contribute to the formation of hyperphosphorylated tau and the pathogenesis of AD.

Asunto(s)

Enfermedad de Alzheimer/metabolismo , Conducta Animal/fisiología , Encéfalo/metabolismo , Aprendizaje por Laberinto/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/psicología , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Encéfalo/patología , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Transgénicos , Neuritas/metabolismo , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Neuronas/patología , Fosforilación , Presenilina-1/genética , Presenilina-1/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas tau/genética

RESUMEN

Two Ebp1 isoproteins, p48 and p42, regulate cell survival and differentiation distinctively. Here we show that p48 is the major isoform in hippocampal neurons and is localized throughout the entire neuron. Notably, reduction of p48 Ebp1 expression inhibited BDNF-mediated neurite outgrowth in hippocampal neurons. The p48 protein acts as a downstream effector of the Trk receptor, which mediates the functions of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in hippocampal cells. Trk receptor activation by both NGF and BDNF induced phosphorylation of Ebp1 at the S360 upon the activation of protein kinase Cδ (PKCδ) and triggered dissociation of p48 from retinoblastoma (Rb). Although both NGF and BDNF activate mitogen-activated protein kinase (MAPK; extracellular signal-related kinase (ERK)) as well as phosphatidylinositide 3-kinase (PI3K)/Akt, their activation is regulated in different time-frame upon growth factor specificity, especially, eliciting PKCδ mediated p48 S360 phosphorylation. Thus, p48 Ebp1 contributes to neuronal cell differentiation and growth factor specificity through the activation of PKCδ, acting as a crucial downstream effector of neurotrophin signaling.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Diferenciación Celular/fisiología , Factores de Crecimiento Nervioso/metabolismo , Neurogénesis/fisiología , Neuronas/metabolismo , Receptor trkA/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Transformada , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Factor de Crecimiento Nervioso/metabolismo , Factor de Crecimiento Nervioso/farmacología , Neurogénesis/efectos de los fármacos , Neuronas/efectos de los fármacos , Células PC12 , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Ratas , Receptor trkA/agonistas , Proteína de Retinoblastoma/metabolismo

RESUMEN

RPS3, a conserved, eukaryotic ribosomal protein of the 40 S subunit, is required for ribosome biogenesis. Because ribosomal proteins are abundant and ubiquitous, they may have additional extraribosomal functions. Here, we show that human RPS3 is a physiological target of Akt kinase and a novel mediator of neuronal apoptosis. NGF stimulation resulted in phosphorylation of threonine 70 of RPS3 by Akt, and this phosphorylation was required for Akt binding to RPS3. RPS3 induced neuronal apoptosis, up-regulating proapoptotic proteins Dp5/Hrk and Bim by binding to E2F1 and acting synergistically with it. Akt-dependent phosphorylation of RPS3 inhibited its proapoptotic function and perturbed its interaction with E2F1. These events coincided with nuclear translocation and accumulation of RPS3, where it functions as an endonuclease. Nuclear accumulation of RPS3 results in an increase in DNA repair activity to some extent, thereby sustaining neuronal survival. Abolishment of Akt-mediated RPS3 phosphorylation through mutagenesis accelerated apoptotic cell death and severely compromised nuclear translocation of RPS3. Thus, our findings define an extraribosomal role of RPS3 as a molecular switch that accommodates apoptotic induction to DNA repair through Akt-mediated phosphorylation.

Asunto(s)

Reparación del ADN/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Ribosómicas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Células Cultivadas , Fragmentación del ADN/efectos de los fármacos , Reparación del ADN/genética , Inmunoprecipitación , Factor de Crecimiento Nervioso/farmacología , Células PC12 , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Proteínas Proto-Oncogénicas c-akt/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Ribosómicas/genética

RESUMEN

Phosphatidylinositol (3,4,5)-triphosphate (PIP(3)) is a lipid second messenger that employs a wide range of downstream effector proteins for the regulation of cellular processes, including cell survival, polarization and proliferation. One of the most well characterized cytoplasmic targets of PIP(3), serine/threonine protein kinase B (PKB)/Akt, promotes cell survival by directly interacting with nucleophosmin (NPM)/B23, the nuclear target of PIP(3). Here, we report that nuclear PIP(3) competes with Akt to preferentially bind B23 in the nucleoplasm. Mutation of Arg23 and Arg25 in the PH domain of Akt prevents binding to PIP(3), but does not disrupt the Akt/B23 interaction. However, treatment with phosphatases PTEN or SHIP abrogates the association between Akt and B23, indicating that nuclear PIP(3) regulates the Akt/B23 interaction by controlling the concentration and subcellular dynamics of these two proteins.

Asunto(s)

Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Fosfatidilinositoles/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Línea Celular Tumoral , Nucleofosmina , Estructura Terciaria de Proteína , Ratas

RESUMEN

Disabled 1 (Dab1), a cytoplasmic adaptor protein expressed predominantly in the CNS, transduces a Reelin-initiated signaling that controls neuronal migration and positioning during brain development. To determine the role of Dab1 in neural stem cell (NSC) differentiation, we established a culture of neurospheres derived from the embryonic forebrain of the Dab1(-/-) mice, yotari. Differentiating Dab1(-/-) neurospheres exhibited a higher expression of GFAP, an astrocytic marker, at the expense of neuronal markers. Under Dab1-deficient condition, the expression of NeuroD, a transcription factor for neuronal differentiation, was decreased and the JAK-STAT pathway was evidently increased during differentiation of NSC, suggesting the possible involvement of Dab1 in astrocyte differentiation via JAK-STAT pathway. Notably, expression of neural and glial markers and the level of JAK-STAT signaling molecules were not changed in differentiating NSC by Reelin treatment, indicating that differentiation of NSC is Reelin-independent. Immunohistochemical analyses showed a decrease in the number of neurons and an increase in the number of GFAP-positive cells in developing yotari brains. Our results suggest that Dab1 participates in the differentiation of NSCs into a specific cell lineage, thereby maintaining a balance between neurogenesis and gliogenesis.

Asunto(s)

Astrocitos/fisiología , Diferenciación Celular/fisiología , Quinasas Janus/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Factores de Transcripción STAT/metabolismo , Células Madre/fisiología , Animales , Astrocitos/citología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Técnicas de Cultivo de Célula , Linaje de la Célula , Células Cultivadas , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Quinasas Janus/genética , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Nestina , Neuronas/citología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteína Reelina , Factores de Transcripción STAT/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Células Madre/citología