RESUMEN
The microtubule motor cytoplasmic dynein contributes to radial migration of newborn pyramidal neurons in the developing neocortex. Here, we show that AMP-activated protein kinase (AMPK) mediates the nucleus-centrosome coupling, a key process for radial neuronal migration that relies on dynein. Depletion of the catalytic subunit of AMPK in migrating neurons impairs this coupling as well as neuronal migration. AMPK shows overlapping subcellular distribution with cytoplasmic dynein and the two proteins interact with each other. Pharmacological inhibition or activation of AMPK modifies the phosphorylation states of dynein intermediate chain (DIC) and dynein functions. Furthermore, AMPK phosphorylates DIC at Ser81. Expression of a phospho-resistant mutant of DIC retards neuronal migration in a similar way to AMPK depletion. Conversely, expression of the phospho-mimetic mutant of DIC alleviates impaired neuronal migration caused by AMPK depletion. Thus, AMPK-regulated dynein function via Ser81 DIC phosphorylation is crucial for radial neuronal migration.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Dineínas Citoplasmáticas/metabolismo , Neocórtex/metabolismo , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/genética , Animales , Movimiento Celular , Núcleo Celular/metabolismo , Centrosoma/metabolismo , Dineínas Citoplasmáticas/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , Ratones , Ratones Endogámicos ICR , Mutagénesis Sitio-Dirigida , Neuronas/citología , Neuronas/metabolismo , Factor de Transcripción PAX6/metabolismo , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
Down syndrome (DS) also known as Trisomy 21 is a genetic disorder that occurs in â¼1 in 800 live births. The disorder is caused by the triplication of all or part of human chromosome 21 and therefore, is thought to arise from the increased dosage of genes found within chromosome 21. The manifestations of the disease include among others physical growth delays and intellectual disability. A prominent anatomical feature of DS is the microcephaly that results from altered brain development. Recent studies using mouse models of DS have shed new light on DYRK1A (dual-specificity tyrosine-phosphorylation-regulated kinase 1A), a gene located on human chromosome 21 that plays a critical role in neocortical development. The present review summarizes effects of the increased dosage of DYRK1A on the proliferative, neurogenic and astrogliogenic potentials of cortical neural progenitor cells, and relates these findings to the clinical manifestations of the disease.
Asunto(s)
Síndrome de Down/fisiopatología , Microcefalia/fisiopatología , Neocórtex/crecimiento & desarrollo , Células-Madre Neurales/fisiología , Neurogénesis/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Quinasas/fisiología , Animales , Síndrome de Down/complicaciones , Humanos , Ratones , Microcefalia/complicaciones , Quinasas DyrKRESUMEN
Newborn neurons in the developing neocortex undergo radial migration, a process that is coupled with their precise passage from multipolar to bipolar shape. The cell-extrinsic signals that govern this transition are, however, poorly understood. Here, we find that lysophosphatidic acid (LPA) signaling contributes to the establishment of a bipolar shape in mouse migratory neurons through LPA receptor 4 (LPA4). LPA4 is robustly expressed in migratory neurons. LPA4-depleted neurons show impaired multipolar-to-bipolar transition and become arrested in their migration. Further, LPA4-mediated LPA signaling promotes formation of the pia-directed process in primary neurons overlaid on neocortical slices. In addition, LPA4 depletion is coupled with altered actin organization as well as with destabilization of the F-actin-binding protein filamin A (FlnA). Finally, overexpression of FlnA rescues the morphology and migration defects of LPA4-depleted neurons. Thus, the LPA-LPA4 axis regulates bipolar morphogenesis and radial migration of newborn cortical neurons via remodeling of the actin cytoskeleton.
Asunto(s)
Movimiento Celular/genética , Polaridad Celular/genética , Lisofosfolípidos/metabolismo , Neocórtex/citología , Neuronas/citología , Receptores Purinérgicos/metabolismo , Células 3T3 , Animales , Línea Celular , Filaminas/metabolismo , Células HEK293 , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Ratones Endogámicos ICR , Neurogénesis/fisiología , Proteínas Nucleares/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptores del Ácido Lisofosfatídico/metabolismo , Receptores Purinérgicos/genética , Proteínas Represoras/metabolismo , Transducción de SeñalRESUMEN
Down syndrome (DS) is caused by trisomy for human chromosome 21. Individuals with DS commonly exhibit mental retardation, which is associated with abnormal brain development. In the neocortex of the DS brain, the density of neurons is markedly reduced, whereas that of astrocytes is increased. Similar to abnormalities seen in DS brains, mouse models of DS show deficits in brain development, and neural progenitor cells that give rise to neurons and glia show dysregulation in their differentiation. These suggest that the dysregulation of progenitor fate choices contributes to alterations in the numbers of neurons and astrocytes in the DS brain. Nevertheless, the molecular basis underlying these defects remains largely unknown. We showed that the overexpression of two human chromosome 21 genes, DYRK1A and DSCR1, contributes to suppressed neuronal differentiation of progenitors in the Ts1Cje mouse model of DS. In addition, the effect of DYRK1A and DSCR1 overexpression on neuronal differentiation is mediated by excessive attenuation of the transcription factor NFATc. Additionally, we demonstrated that an increased dosage of DYRK1A contributes to elevated potential of Ts1Cje progenitors to differentiate into astrocytes and enhanced astrogliogenesis in the Ts1Cje neocortex. Further, we linked the increased dosage of DYRK1A to dysregulation of STAT, a transcription factor critical for astrogliogenesis. Together, our studies identify critical pathways responsible for the proper differentiation of neural progenitors into neurons and astrocytes, with direct implications for the anomalies in brain development observed in DS.
Asunto(s)
Encéfalo/citología , Encéfalo/patología , Diferenciación Celular/efectos de los fármacos , Síndrome de Down/genética , Síndrome de Down/patología , Células-Madre Neurales/citología , Neurogénesis/genética , Animales , Astrocitos , Cromosomas Humanos Par 21/genética , Proteínas de Unión al ADN , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/fisiología , Ratones , Proteínas Musculares/genética , Proteínas Musculares/fisiología , Factores de Transcripción NFATC , Neuroglía , Neuronas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/fisiología , Factores de Transcripción STAT , Quinasas DyrKRESUMEN
Nuclear distribution element-like 1 (Ndel1) plays pivotal roles in diverse biological processes and is implicated in the pathogenesis of multiple neurodevelopmental disorders. Ndel1 function by regulating microtubules and intermediate filaments; however, its functional link with the actin cytoskeleton is largely unknown. Here, we show that Ndel1 interacts with TRIO-associated repeat on actin (Tara), an actin-bundling protein, to regulate cell movement. In vitro wound healing and Boyden chamber assays revealed that Ndel1- or Tara-deficient cells were defective in cell migration. Moreover, Tara overexpression induced the accumulation of Ndel1 at the cell periphery and resulted in prominent co-localization with F-actin. This redistribution of Ndel1 was abolished by deletion of the Ndel1-interacting domain of Tara, suggesting that the altered peripheral localization of Ndel1 requires a physical interaction with Tara. Furthermore, co-expression of Ndel1 and Tara in SH-SY5Y cells caused a synergistic increase in F-actin levels and filopodia formation, suggesting that Tara facilitates cell movement by sequestering Ndel1 at peripheral structures to regulate actin remodeling. Thus, we demonstrated that Ndel1 interacts with Tara to regulate cell movement. These findings reveal a novel role of the Ndel1-Tara complex in actin reorganization during cell movement.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Microfilamentos/metabolismo , Actinas/metabolismo , Proteínas Portadoras/genética , Línea Celular , Movimiento Celular , Eliminación de Gen , Humanos , Proteínas de Microfilamentos/genéticaRESUMEN
The ability of radial glial progenitors (RGPs) to generate cortical neurons is determined by local extracellular factors and signaling pathways intrinsic to RGPs. Here we find that GPR157, an orphan G protein-coupled receptor, localizes to RGPs' primary cilia exposed to the cerebrospinal fluid (CSF). GPR157 couples with Gq-class of the heterotrimeric G-proteins and signals through IP3-mediated Ca(2+) cascade. Activation of GPR157-Gq signaling enhances neuronal differentiation of RGPs whereas interfering with GPR157-Gq-IP3 cascade in RGPs suppresses neurogenesis. We also detect the presence of putative ligand(s) for GPR157 in the CSF, and demonstrate the increased ability of the CSF to activate GPR157 at neurogenic phase. Thus, GPR157-Gq signaling at the primary cilia of RGPs is activated by the CSF and contributes to neurogenesis.
Asunto(s)
Señalización del Calcio , Diferenciación Celular , Células Ependimogliales/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Neuronas/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Células Madre/fisiología , Animales , Líquido Cefalorraquídeo/metabolismo , Cilios/química , Ratones Endogámicos ICR , Neurogénesis , Células Madre/químicaRESUMEN
Down syndrome (DS) arises from triplication of genes on human chromosome 21 and is associated with anomalies in brain development such as reduced production of neurons and increased generation of astrocytes. Here, we show that differentiation of cortical progenitor cells into astrocytes is promoted by DYRK1A, a Ser/Thr kinase encoded on human chromosome 21. In the Ts1Cje mouse model of DS, increased dosage of DYRK1A augments the propensity of progenitors to differentiate into astrocytes. This tendency is associated with enhanced astrogliogenesis in the developing neocortex. We also find that overexpression of DYRK1A upregulates the activity of the astrogliogenic transcription factor STAT in wild-type progenitors. Ts1Cje progenitors exhibit elevated STAT activity, and depletion of DYRK1A in these cells reverses the deregulation of STAT. In sum, our findings indicate that potentiation of the DYRK1A-STAT pathway in progenitors contributes to aberrant astrogliogenesis in DS.
Asunto(s)
Astrocitos/citología , Síndrome de Down/fisiopatología , Neocórtex/fisiopatología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Factores de Transcripción STAT/metabolismo , Animales , Astrocitos/fisiología , Diferenciación Celular , Síndrome de Down/genética , Síndrome de Down/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Masculino , Ratones , Neocórtex/patología , Células Madre/fisiología , Quinasas DyrKRESUMEN
The human ortholog of the targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a cytoskeletal protein that plays a major role in spindle assembly and is required for mitosis. During spindle morphogenesis, TPX2 cooperates with Aurora A kinase and Eg5 kinesin to regulate microtubule organization. TPX2 displays over 40 putative phosphorylation sites identified from various high-throughput proteomic screenings. In this study, we characterize the phosphorylation of threonine 72 (Thr(72)) in human TPX2, a residue highly conserved across species. We find that Cdk1/2 phosphorylate TPX2 in vitro and in vivo. Using homemade antibodies specific for TPX2 phosphorylated at Thr(72), we show that this phosphorylation is cell cycle-dependent and peaks at M phase. Endogenous TPX2 phosphorylated at Thr(72) does not associate with the mitotic spindle. Furthermore, ectopic GFP-TPX2 T72A preferentially concentrates on the spindle, whereas GFP-TPX2 WT distributes to both spindle and cytosol. The T72A mutant also increases the proportion of cells with multipolar spindles phenotype. This effect is associated with increased Aurora A activity and abnormally elongated spindles, indicative of higher Eg5 activity. In summary, we propose that phosphorylation of Thr(72) regulates TPX2 localization and impacts spindle assembly via Aurora A and Eg5.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Huso Acromático , Treonina/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Secuencia de Bases , Proteínas de Ciclo Celular/química , Cartilla de ADN , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/química , Proteínas Nucleares/química , Fosfoproteínas/química , Fosforilación , Treonina/química , Xenopus , Proteínas de Xenopus/químicaRESUMEN
The large microtubule-associated/Ca(2+)-signalling protein p600 (also known as UBR4) is required for hippocampal neuronal survival upon Ca(2+) dyshomeostasis induced by glutamate treatment. During this process, p600 prevents aggregation of the Ca(2+)/calmodulin-dependent kinase IIα (CaMKIIα), a proxy of neuronal death, via direct binding to calmodulin in a microtubuleindependent manner. Using photoconductive stimulation coupled with live imaging of single neurons, we identified a distinct mechanism of prevention of CaMKIIα aggregation by p600. Upon direct depolarization, CaMKIIα translocates to microtubules. In the absence of p600, this translocation is interrupted in favour of a sustained self-aggregation that is prevented by the microtubule-stabilizing drug paclitaxel. Thus, during photoconductive stimulation, p600 prevents the aggregation of CaMKIIα by stabilizing microtubules. The effectiveness of this stabilization for preventing CaMKIIα aggregation during direct depolarization but not during glutamate treatment suggests a model wherein p600 has two modes of action depending on the source of cytosolic Ca(2+).
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuronas/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Células Cultivadas , Dendritas/metabolismo , Hipocampo/citología , Luz , Proteínas Asociadas a Microtúbulos/genética , Neuronas/citología , Neuronas/efectos de la radiación , Interferencia de ARN , Ratas , Análisis de la Célula Individual/métodos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Apical neural progenitors (aNPs) drive neurogenesis by means of a program consisting of self-proliferative and neurogenic divisions. The balance between these two manners of division sustains the pool of apical progenitors into late neurogenesis, thereby ensuring their availability to populate the brain with terminal cell types. Using knockout and in utero electroporation mouse models, we report a key role for the microtubule-associated protein 600 (p600) in the regulation of spindle orientation in aNPs, a cellular event that has been associated with cell fate and neurogenesis. We find that p600 interacts directly with the neurogenic protein Ndel1 and that aNPs knockout for p600, depleted of p600 by shRNA or expressing a Ndel1-binding p600 fragment all display randomized spindle orientation. Depletion of p600 by shRNA or expression of the Ndel1-binding p600 fragment also results in a decreased number of Pax6-positive aNPs and an increased number of Tbr2-positive basal progenitors destined to become neurons. These Pax6-positive aNPs display a tilted mitotic spindle. In mice wherein p600 is ablated in progenitors, the production of neurons is significantly impaired and this defect is associated with microcephaly. We propose a working model in which p600 controls spindle orientation in aNPs and discuss its implication for neurogenesis.
RESUMEN
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of motor neurons. Here we show that the basic leucine zipper transcription factor NFIL3 (also called E4BP4) confers neuroprotection in models of ALS. NFIL3 is up-regulated in primary neurons challenged with neurotoxic insults and in a mouse model of ALS. Overexpression of NFIL3 attenuates excitotoxic neuronal damage and protects neurons against neurodegeneration in a cell-based ALS model. Conversely, reduction of NFIL3 exacerbates neuronal demise in adverse conditions. Transgenic neuronal expression of NFIL3 in ALS mice delays disease onset and attenuates motor axon and neuron degeneration. These results suggest that NFIL3 plays a neuroprotective role in neurons and constitutes a potential therapeutic target for neurodegeneration.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Axones/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Neuronas Motoras/metabolismo , Fármacos Neuroprotectores/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Axones/patología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Neuronas Motoras/patologíaRESUMEN
Down's syndrome (DS), a major genetic cause of mental retardation, arises from triplication of genes on human chromosome 21. Here we show that DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) and DSCR1 (DS critical region 1), two genes lying within human chromosome 21 and encoding for a serine/threonine kinase and calcineurin regulator, respectively, are expressed in neural progenitors in the mouse developing neocortex. Increasing the dosage of both proteins in neural progenitors leads to a delay in neuronal differentiation, resulting ultimately in alteration of their laminar fate. This defect is mediated by the cooperative actions of DYRK1A and DSCR1 in suppressing the activity of the transcription factor NFATc. In Ts1Cje mice, a DS mouse model, dysregulation of NFATc in conjunction with increased levels of DYRK1A and DSCR1 was observed. Furthermore, counteracting the dysregulated pathway ameliorates the delayed neuronal differentiation observed in Ts1Cje mice. In sum, our findings suggest that dosage of DYRK1A and DSCR1 is critical for proper neurogenesis through NFATc and provide a potential mechanism to explain the neurodevelopmental defects in DS.
Asunto(s)
Diferenciación Celular/genética , Dosificación de Gen/genética , Péptidos y Proteínas de Señalización Intracelular , Proteínas Musculares , Neocórtex/citología , Neurogénesis/genética , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas , Células Madre/citología , Animales , Proteínas de Unión al Calcio , Modelos Animales de Enfermedad , Síndrome de Down/genética , Síndrome de Down/patología , Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Factores de Transcripción NFATC/metabolismo , Neocórtex/embriología , Plásmidos/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Quinasas DyrKRESUMEN
Neural progenitor cells in the developing brain give rise to neurons and glia. Multiple extrinsic signalling molecules and their cognate membrane receptors have been identified to control neural progenitor fate. However, a role for G protein-coupled receptors in cell fate decisions in the brain remains largely putative. Here we show that GPRC5B, which encodes an orphan G protein-coupled receptor, is present in the ventricular surface of cortical progenitors in the mouse developing neocortex and is required for their neuronal differentiation. GPRC5B-depleted progenitors fail to adopt a neuronal fate and ultimately become astrocytes. Furthermore, GPRC5B-mediated signalling is associated with the proper regulation of ß-catenin signalling, a pathway crucial for progenitor fate decision. Our study uncovers G protein-coupled receptor signalling in the neuronal fate determination of cortical progenitors.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Neocórtex/embriología , Células-Madre Neurales/fisiología , Neurogénesis/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , Animales , Diferenciación Celular/genética , Cartilla de ADN/genética , Electroporación , Regulación del Desarrollo de la Expresión Génica/genética , Inmunohistoquímica , Hibridación in Situ , Ratones , Neocórtex/metabolismo , Neurogénesis/genética , Plásmidos/genéticaRESUMEN
In acute and chronic neurodegeneration, Ca(2+) mishandling and disruption of the cytoskeleton compromise neuronal integrity, yet abnormalities in the signaling roles of cytoskeletal proteins remain largely unexplored. We now report that the microtubule-associated protein p600 (also known as UBR4) promotes neuronal survival. Following depletion of p600, glutamate-induced Ca(2+) influx through NMDA receptors, but not AMPA receptors, initiates a degenerative process characterized by endoplasmic reticulum fragmentation and endoplasmic reticulum Ca(2+) release via inositol 1,4,5-trisphosphate receptors. Downstream of NMDA receptors, p600 associates with the calmodulin·calmodulin-dependent protein kinase IIα complex. A direct and atypical p600/calmodulin interaction is required for neuronal survival. Thus, p600 counteracts specific Ca(2+)-induced death pathways through regulation of Ca(2+) homeostasis and signaling.
Asunto(s)
Calcio/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Transducción de Señal/fisiología , Animales , Proteínas de Unión a Calmodulina/genética , Supervivencia Celular/fisiología , Células Cultivadas , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Ratas , Receptores AMPA/genética , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
The maternal care that offspring receive from their mothers early in life influences the offspring's development of emotional behavior in adulthood. Here we found that offspring reared by circadian clock-impaired mice show elevated anxiety-related behavior. Clock mutant mice harboring a mutation in Clock, a key component of the molecular circadian clock, display altered daily patterns of nursing behavior that is fragmented during the light period, instead of long bouts of nursing behavior in wild-type mice. Adult wild-type offspring fostered by Clock mutant mice exhibit increased anxiety-related behavior. This is coupled with reduced levels of brain serotonin at postnatal day 14, whose homeostasis during the early postnatal period is critical for normal emotional behavior in adulthood. Together, disruption of the circadian clock in mothers has an adverse impact on establishing normal anxiety levels in offspring, which may increase their risk of developing anxiety disorders.
Asunto(s)
Ansiedad/etiología , Proteínas CLOCK/genética , Conducta Materna/fisiología , Animales , Encéfalo/metabolismo , Proteínas CLOCK/fisiología , Femenino , Ratones , Mutación/genética , Serotonina/metabolismoRESUMEN
Neuronal migration is an essential process for the development of the cerebral cortex. We have previously shown that LKB1, an evolutionally conserved polarity kinase, plays a critical role in neuronal migration in the developing neocortex. Here we show that LKB1 mediates Ser9 phosphorylation of GSK3beta to inactivate the kinase at the leading process tip of migrating neurons in the developing neocortex. This enables the microtubule plus-end binding protein adenomatous polyposis coli (APC) to localize at the distal ends of microtubules in the tip, thereby stabilizing microtubules near the leading edge. We also show that LKB1 activity, Ser9 phosphorylation of GSK3beta, and APC binding to the distal ends of microtubules are required for the microtubule stabilization in the leading process tip, centrosomal forward movement, and neuronal migration. These findings suggest that LKB1-induced spatial control of GSK3beta and APC at the leading process tip mediates the stabilization of microtubules within the tip and is critical for centrosomal forward movement and neuronal migration in the developing neocortex.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Centrosoma/fisiología , Glucógeno Sintasa Quinasa 3/metabolismo , Neocórtex/crecimiento & desarrollo , Neuronas/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Secuencia de Aminoácidos , Animales , Línea Celular , Movimiento Celular/fisiología , Células Cultivadas , Técnicas de Silenciamiento del Gen , Glucógeno Sintasa Quinasa 3/química , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Humanos , Técnicas In Vitro , Ratones , Microtúbulos/metabolismo , Neocórtex/fisiología , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Serina/metabolismoRESUMEN
Circadian molecular oscillation is generated by a transcription/translation-based feedback loop in which CRY proteins play critical roles as potent inhibitors for E-box-dependent clock gene expression. Although CRY2 undergoes rhythmic phosphorylation in its C-terminal tail, structurally distinct from the CRY1 tail, little is understood about how protein kinase(s) controls the CRY2-specific phosphorylation and contributes to the molecular clockwork. Here we found that Ser557 in the C-terminal tail of CRY2 is phosphorylated by DYRK1A as a priming kinase for subsequent GSK-3beta (glycogen synthase kinase 3beta)-mediated phosphorylation of Ser553, which leads to proteasomal degradation of CRY2. In the mouse liver, DYRK1A kinase activity toward Ser557 of CRY2 showed circadian variation, with its peak in the accumulating phase of CRY2 protein. Knockdown of Dyrk1a caused abnormal accumulation of cytosolic CRY2, advancing the timing of a nuclear increase of CRY2, and shortened the period length of the cellular circadian rhythm. Expression of an S557A/S553A mutant of CRY2 phenocopied the effect of Dyrk1a knockdown in terms of the circadian period length of the cellular clock. DYRK1A is a novel clock component cooperating with GSK-3beta and governs the Ser557 phosphorylation-triggered degradation of CRY2.
Asunto(s)
Ritmo Circadiano/fisiología , Criptocromos/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Relojes Biológicos/fisiología , Células Cultivadas , Criptocromos/genética , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Serina/metabolismo , Quinasas DyrKRESUMEN
BACKGROUND: Adult neurogenesis occurs in specific regions of the mammalian brain such as the dentate gyrus of the hippocampus. In the neurogenic region, neural progenitor cells continuously divide and give birth to new neurons. Although biological properties of neurons and glia in the hippocampus have been demonstrated to fluctuate depending on specific times of the day, it is unclear if neural progenitors and neurogenesis in the adult brain are temporally controlled within the day. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that in the dentate gyrus of the adult mouse hippocampus, the number of M-phase cells shows a day/night variation throughout the day, with a significant increase during the nighttime. The M-phase cell number is constant throughout the day in the subventricular zone of the forebrain, another site of adult neurogenesis, indicating the daily rhythm of progenitor mitosis is region-specific. Importantly, the nighttime enhancement of hippocampal progenitor mitosis is accompanied by a nighttime increase of newborn neurons. CONCLUSIONS/SIGNIFICANCE: These results indicate that neurogenesis in the adult hippocampus occurs in a time-of-day-dependent fashion, which may dictate daily modifications of dentate gyrus physiology.
Asunto(s)
Hipocampo/metabolismo , Neurogénesis/fisiología , Animales , División Celular , Giro Dentado/fisiología , Inmunohistoquímica , Ratones , Modelos Biológicos , Factores de TiempoRESUMEN
There is an increasing body of literature pointing to cytoskeletal proteins as spatial organizers and interactors of organelles. In this study, we identified protein 600 (p600) as a novel microtubule-associated protein (MAP) developmentally regulated in neurons. p600 exhibits the unique feature to interact with the endoplasmic reticulum (ER). Silencing of p600 by RNA interference (RNAi) destabilizes neuronal processes in young primary neurons undergoing neurite extension and containing scarce staining of the ER marker Bip. Furthermore, in utero electroporation of p600 RNAi alters neuronal migration, a process that depends on synergistic actions of microtubule dynamics and ER functions. p600-depleted migrating neurons display thin, crooked, and "zigzag" leading process with very few ER membranes. Thus, p600 constitutes the only known MAP to associate with the ER in neurons, and this interaction may impact on multiple cellular processes ranging from neuronal development to neuronal maturation and plasticity.
Asunto(s)
Sistema Nervioso Central/citología , Retículo Endoplásmico/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/metabolismo , Neuronas/ultraestructura , Animales , Animales Recién Nacidos , Proteínas de Unión a Calmodulina , Diferenciación Celular/fisiología , Células Cultivadas , Chaperón BiP del Retículo Endoplásmico , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Choque Térmico/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Inmunoelectrónica/métodos , Proteínas Asociadas a Microtúbulos/genética , Chaperonas Moleculares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuritas/metabolismo , Proteínas de Neurofilamentos/deficiencia , Interferencia de ARN/fisiología , Transfección/métodos , Tubulina (Proteína)/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Centrosome- and microtubule-associated proteins have been shown to be important for maintaining the neural progenitor pool during neocortical development by regulating the mitotic spindle. It remains unclear whether these proteins may control neurogenesis by regulating other microtubule-dependent processes such as nuclear migration. Here, we identify Cep120, a centrosomal protein preferentially expressed in neural progenitors during neocortical development. We demonstrate that silencing Cep120 in the developing neocortex impairs both interkinetic nuclear migration (INM), a characteristic pattern of nuclear movement in neural progenitors, and neural progenitor self-renewal. Furthermore, we show that Cep120 interacts with transforming acidic coiled-coil proteins (TACCs) and that silencing TACCs also causes defects in INM and neural progenitor self-renewal. Our data suggest a critical role for Cep120 and TACCs in both INM and neurogenesis. We propose that sustaining INM may be a mechanism by which microtubule-regulating proteins maintain the neural progenitor pool during neocortical development.