RESUMO
Cell differentiation involves profound changes in global gene expression that often has to occur in coordination with cell cycle exit. Because cyclin-dependent kinase inhibitor p27 reportedly regulates proliferation of neural progenitor cells in the subependymal neurogenic niche of the adult mouse brain, but can also have effects on gene expression, we decided to molecularly analyze its role in adult neurogenesis and oligodendrogenesis. At the cell level, we show that p27 restricts residual cyclin-dependent kinase activity after mitogen withdrawal to antagonize cycling, but it is not essential for cell cycle exit. By integrating genome-wide gene expression and chromatin accessibility data, we find that p27 is coincidentally necessary to repress many genes involved in the transit from multipotentiality to differentiation, including those coding for neural progenitor transcription factors SOX2, OLIG2 and ASCL1. Our data reveal both a direct association of p27 with regulatory sequences in the three genes and an additional hierarchical relationship where p27 repression of Sox2 leads to reduced levels of its downstream targets Olig2 and Ascl1. In vivo, p27 is also required for the regulation of the proper level of SOX2 necessary for neuroblasts and oligodendroglial progenitor cells to timely exit cell cycle in a lineage-dependent manner.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27 , Neurogênese , Fatores de Transcrição SOXB1 , Animais , Camundongos , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Divisão Celular , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Expressão Gênica , Neurogênese/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
Several psychiatric, neurologic and neurodegenerative disorders present increased brain ventricles volume, being hydrocephalus the disease with the major manifestation of ventriculomegaly caused by the accumulation of high amounts of cerebrospinal fluid (CSF). The molecules and pathomechanisms underlying cerebral ventricular enlargement are widely unknown. Kinase D interacting substrate of 220 kDa (KIDINS220) gene has been recently associated with schizophrenia and with a novel syndrome characterized by spastic paraplegia, intellectual disability, nystagmus and obesity (SINO syndrome), diseases frequently occurring with ventriculomegaly. Here we show that Kidins220, a transmembrane protein effector of various key neuronal signalling pathways, is a critical regulator of CSF homeostasis. We observe that both KIDINS220 and the water channel aquaporin-4 (AQP4) are markedly downregulated at the ventricular ependymal lining of idiopathic normal pressure hydrocephalus (iNPH) patients. We also find that Kidins220 deficient mice develop ventriculomegaly accompanied by water dyshomeostasis and loss of AQP4 in the brain ventricular ependymal layer and astrocytes. Kidins220 is a known cargo of the SNX27-retromer, a complex that redirects endocytosed plasma membrane proteins (cargos) back to the cell surface, thus avoiding their targeting to lysosomes for degradation. Mechanistically, we show that AQP4 is a novel cargo of the SNX27-retromer and that Kidins220 deficiency promotes a striking and unexpected downregulation of the SNX27-retromer that results in AQP4 lysosomal degradation. Accordingly, SNX27 silencing decreases AQP4 levels in wild-type astrocytes whereas SNX27 overexpression restores AQP4 content in Kidins220 deficient astrocytes. Together our data suggest that the KIDINS220-SNX27-retromer-AQP4 pathway is involved in human ventriculomegaly and open novel therapeutic perspectives.
Assuntos
Hidrocefalia , Animais , Aquaporina 4/genética , Aquaporina 4/metabolismo , Epêndima/metabolismo , Humanos , Hidrocefalia/genética , Hidrocefalia/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nexinas de Classificação/genéticaRESUMO
We used proximity-dependent biotin identification (BioID) to find proteins that potentially interact with the major glial glutamate transporter, GLT-1, and we studied how these interactions might affect its activity. GTPase Rac1 was one protein identified, and interfering with its GTP/GDP cycle in mixed primary rat brain cultures affected both the clustering of GLT-1 at the astrocytic processes and the transport kinetics, increasing its uptake activity at low micromolar glutamate concentrations in a manner that was dependent on the effector kinase PAK1 and the actin cytoskeleton. Interestingly, the same manipulations had a different effect on another glial glutamate transporter, GLAST, inhibiting its activity. Importantly, glutamate acts through metabotropic receptors to stimulate the activity of Rac1 in astrocytes, supporting the existence of cross-talk between extracellular glutamate and the astrocytic form of the GLT-1 regulated by Rac1. CDC42EP4/BORG4 (a CDC42 effector) was also identified in the BioID screen, and it is a protein that regulates the assembly of septins and actin fibers, influencing the organization of the cytoskeleton. We found that GLT-1 interacts with septins, which reduces its lateral mobility at the cell surface. Finally, the G-protein subunit GNB4 dampens the activity of GLT-1, as revealed by its response to the activator peptide mSIRK, both in heterologous systems and in primary brain cultures. This effect occurs rapidly and thus, it is unlikely to depend on cytoskeletal dynamics. These novel interactions shed new light on the events controlling GLT-1 activity, thereby helping us to better understand how glutamate homeostasis is maintained in the brain.
Assuntos
Transporte Biológico/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Neuroglia/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Biotinilação , Células COS , Células Cultivadas , Córtex Cerebral/citologia , Chlorocebus aethiops , Embrião de Mamíferos , Transportador 2 de Aminoácido Excitatório/genética , Ácido Glutâmico , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Células HEK293 , Humanos , Imunoprecipitação , Modelos Moleculares , Neuroglia/efeitos dos fármacos , Neurônios/metabolismo , Fotodegradação , Ratos , TransfecçãoRESUMO
Stem cells maintain their self-renewal and multipotency capacities through a self-organizing network of transcription factors and intracellular pathways activated by extracellular signaling from the microenvironment or "niche" in which they reside in vivo. In the adult mammalian brain new neurons continue to be generated throughout life of the organisms and this lifelong process of neurogenesis is supported by a reservoir of neural stem cells in the germinal regions. The discovery of adult neurogenesis in the mammalian brain has sparked great interest in defining the conditions that guide neural stem cell (NSC) maintenance and differentiation into the great variety of neuronal and glial subtypes. Here we review current knowledge regarding the paracrine regulation provided by the components of the niche and its function, focusing on the main germinal region of the adult central nervous system (CNS), the subependymal zone (SEZ).
Assuntos
Movimento Celular , Epêndima/fisiologia , Células-Tronco Neurais/fisiologia , Comunicação Parácrina , Animais , Astrócitos/metabolismo , Astrócitos/fisiologia , Proliferação de Células , Plexo Corióideo/metabolismo , Plexo Corióideo/fisiologia , Epêndima/metabolismo , Humanos , Neovascularização Fisiológica , Células-Tronco Neurais/metabolismo , Neurogênese , Bulbo Olfatório/metabolismo , Bulbo Olfatório/fisiologia , Nicho de Células-Tronco , Transmissão SinápticaRESUMO
In the adult mammalian brain, neural stem cells (NSCs) located in highly restricted niches sustain the generation of new neurons that integrate into existing circuits. A reduction in adult neurogenesis is linked to ageing and neurodegeneration, whereas dysregulation of proliferation and survival of NSCs have been hypothesized to be at the origin of glioma. Thus, unravelling the molecular underpinnings of the regulated activation that NSCs must undergo to proliferate and generate new progeny is of considerable relevance. Current research has identified cues promoting or restraining NSCs activation. Yet, whether NSCs depend on external signals to survive or if intrinsic factors establish a threshold for sustaining their viability remains elusive, even if this knowledge could involve potential for devising novel therapeutic strategies. Kidins220 (Kinase D-interacting substrate of 220 kDa) is an essential effector of crucial pathways for neuronal survival and differentiation. It is dramatically altered in cancer and in neurological and neurodegenerative disorders, emerging as a regulatory molecule with important functions in human disease. Herein, we discover severe neurogenic deficits and hippocampal-based spatial memory defects accompanied by increased neuroblast death and high loss of newly formed neurons in Kidins220 deficient mice. Mechanistically, we demonstrate that Kidins220-dependent activation of AKT in response to EGF restraints GSK3 activity preventing NSCs apoptosis. We also show that NSCs with Kidins220 can survive with lower concentrations of EGF than the ones lacking this molecule. Hence, Kidins220 levels set a molecular threshold for survival in response to mitogens, allowing adult NSCs growth and expansion. Our study identifies Kidins220 as a key player for sensing the availability of growth factors to sustain adult neurogenesis, uncovering a molecular link that may help paving the way towards neurorepair.
Assuntos
Células-Tronco Adultas , Células-Tronco Neurais , Adulto , Animais , Humanos , Camundongos , Células-Tronco Adultas/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Hipocampo/metabolismo , Mamíferos , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismoRESUMO
Alteration of centrosome function and dynamics results in major defects during chromosome segregation and is associated with primary autosomal microcephaly (MCPH). Despite the knowledge accumulated in the last few years, why some centrosomal defects specifically affect neural progenitors is not clear. We describe here that the centrosomal kinase PLK1 controls centrosome asymmetry and cell fate in neural progenitors during development. Gain- or loss-of-function mutations in Plk1, as well as deficiencies in the MCPH genes Cdk5rap2 (MCPH3) and Cep135 (MCPH8), lead to abnormal asymmetry in the centrosomes carrying the mother and daughter centriole in neural progenitors. However, whereas loss of MCPH proteins leads to increased centrosome asymmetry and microcephaly, deficient PLK1 activity results in reduced asymmetry and increased expansion of neural progenitors and cortical growth during mid-gestation. The combination of PLK1 and MCPH mutations results in increased microcephaly accompanied by more aggressive centrosomal and mitotic abnormalities. In addition to highlighting the delicate balance in the level and activity of centrosomal regulators, these data suggest that human PLK1, which maps to 16p12.1, may contribute to the neurodevelopmental defects associated with 16p11.2-p12.2 microdeletions and microduplications in children with developmental delay and dysmorphic features.
Assuntos
Proteínas de Ciclo Celular , Microcefalia , Células-Tronco Neurais , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas , Proteínas de Ciclo Celular/genética , Diferenciação Celular , Centrossomo/metabolismo , Criança , Segregação de Cromossomos , Humanos , Microcefalia/genética , Microcefalia/metabolismo , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Quinase 1 Polo-LikeRESUMO
In the mammalian adult brain, neural stem cells persist in neurogenic niches. The subependymal zone is the most prolific neurogenic niche in adult rodents, where residing stem cells generate large numbers of immature neurons that migrate into the olfactory bulb, where they differentiate into different types of interneurons. Subependymal neural stem cells derive from embryonic radial glia and retain some of their features like apico-basal polarity, with apical processes piercing the ependymal layer, and a basal process contacting blood vessels, constituting an epithelial niche. Conservation of the cytoarchitecture of the niche is of crucial importance for the maintenance of stem cells and for their neurogenic potential. In this minireview we will focus on extracellular matrix and adhesion molecules in the adult subependymal zone, showing their involvement not only as structural elements sustaining the niche architecture and topology, but also in the maintenance of stemness and regulation of the quiescence-proliferation balance.
RESUMO
Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos , Lentivirus/genética , Células-Tronco Neurais/citologia , Transfecção/métodos , Animais , Encéfalo/patologia , Epêndima/citologia , Ventrículos Laterais/citologia , Ventrículos Laterais/metabolismo , CamundongosRESUMO
Thyroid hormone receptors (TRs) are members of the ligand-inducible transcription factor superfamily. The two major functional TRs (alpha1 and beta1) have different spatial and temporal expression patterns and specific physiological functions for these isoforms are now starting to emerge. By expressing these TR isoforms individually in Swiss 3T3 fibroblasts, we found that TRbeta1 expression, in the absence of hormone, provokes a proliferation arrest in G0/G1, lengthening the cycling time. Upon serum stimulation TRbeta1-expressing cells showed a marked delay in the induction of cyclins D and E, in the phosphorylation of retinoblastoma protein, and in the activation of cyclin-dependent kinase 2, accompanied by increased levels of cyclin-dependent kinase inhibitor p27Kip1. Accordingly, serum-stimulated E2F-1 transcriptional activity was repressed by TRbeta1 in transient transfection experiments. Analysis of the receptor domains required for this effect confirmed that there is no need for a functional ligand-binding domain while the DNA-binding domain is essential. In this work, we demonstrate for the first time that TRbeta1 participates in the molecular mechanisms that control cell proliferation. The unliganded TRbeta1 impairs the normal induction of the G1/S cycle regulators preventing progression into the S phase.
Assuntos
Divisão Celular/fisiologia , Fase G1 , Receptores beta dos Hormônios Tireóideos/fisiologia , Animais , Sequência de Bases , Western Blotting , Primers do DNA , Fibroblastos/citologia , Citometria de Fluxo , Imunoprecipitação , Ligantes , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Swiss 3T3RESUMO
Interactions of adult neural stem cells (NSCs) with supportive vasculature appear critical for their maintenance and function, although the molecular details are still under investigation. Neurotrophin (NT)-3 belongs to the NT family of trophic factors, best known for their effects in promoting neuronal survival. Here we show that NT-3 produced and secreted by endothelial cells of brain and choroid plexus capillaries is required for the quiescence and long-term maintenance of NSCs in the mouse subependymal niche. Uptake of NT-3 from irrigating vasculature and cerebrospinal fluid (CSF) induces the rapid phosphorylation of endothelial nitric oxide (NO) synthase present in the NSCs, leading to the production of NO, which subsequently acts as a cytostatic factor. Our results identify a novel interaction between stem cells and vasculature/CSF compartments that is mediated by an unprecedented role of a neurotrophin and indicate that stem cells can regulate their own quiescence in response to endothelium-secreted molecules.
Assuntos
Células Endoteliais/metabolismo , Células-Tronco Neurais/citologia , Neurônios/citologia , Neurotrofina 3/metabolismo , Óxido Nítrico/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Camundongos , Óxido Nítrico/líquido cefalorraquidiano , Óxido Nítrico Sintase Tipo III/metabolismoRESUMO
The identification of mechanisms that maintain stem cell niche architecture and homeostasis is fundamental to our understanding of tissue renewal and repair. Cell adhesion is a well-characterized mechanism for developmental morphogenetic processes, but its contribution to the dynamic regulation of adult mammalian stem cell niches is still poorly defined. We show that N-cadherin-mediated anchorage of neural stem cells (NSCs) to ependymocytes in the adult murine subependymal zone modulates their quiescence. We further identify MT5-MMP as a membrane-type metalloproteinase responsible for the shedding of the N-cadherin ectodomain in this niche. MT5-MMP is co-expressed with N-cadherin in adult NSCs and ependymocytes and, whereas MT5-MMP-mediated cleavage of N-cadherin is dispensable for the regulation of NSC generation and identity, it is required for proper activation of NSCs under physiological and regenerative conditions. Our results indicate that the proliferative status of stem cells can be dynamically modulated by regulated cleavage of cell adhesion molecules.
Assuntos
Caderinas/metabolismo , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/enzimologia , Animais , Linfócitos B/metabolismo , Adesão Celular , Proliferação de Células , Células Cultivadas , Imuno-Histoquímica , Camundongos , Fragmentos de Peptídeos/metabolismoRESUMO
The E3-ubiquitin ligase APC/C-Cdh1 is essential for endoreduplication but its relevance in the mammalian mitotic cell cycle is still unclear. Here we show that genetic ablation of Cdh1 in the developing nervous system results in hypoplastic brain and hydrocephalus. These defects correlate with enhanced levels of Cdh1 substrates and increased entry into the S phase in neural progenitors. However, cell division is prevented in the absence of Cdh1 due to hyperactivation of cyclin-dependent kinases, replicative stress, induction of p53, G2 arrest and apoptotic death of these progenitor cells. Concomitant ablation of p53 rescues apoptosis but not replicative stress, resulting in the presence of damaged neurons throughout the adult brain. These data indicate that the inactivation of Cdh1 in vivo results in replicative stress, cell cycle arrest and cell death, supporting recent therapeutic proposals aimed to inhibit the APC/C in tumours.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Apoptose , Encéfalo/metabolismo , Proteínas Cdh1/metabolismo , Replicação do DNA , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Animais , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/enzimologia , Proteínas Cdh1/genética , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/citologia , Células-Tronco Neurais/enzimologia , Neurogênese , Neurônios/citologia , Neurônios/enzimologia , Tamanho do Órgão , Proteína Supressora de Tumor p53/genéticaRESUMO
Relative quiescence and self renewal are defining features of adult stem cells, but their potential coordination remains unclear. Subependymal neural stem cells (NSCs) lacking cyclin-dependent kinase (CDK) inhibitor (CKI) 1a (p21) exhibit rapid expansion that is followed by their permanent loss later in life. Here we demonstrate that transcription of the gene encoding bone morphogenetic protein 2 (Bmp2) in NSCs is under the direct negative control of p21 through actions that are independent of CDK. Loss of p21 in NSCs results in increased levels of secreted BMP2, which induce premature terminal differentiation of multipotent NSCs into mature non-neurogenic astrocytes in an autocrine and/or paracrine manner. We also show that the cell-nonautonomous p21-null phenotype is modulated by the Noggin-rich environment of the subependymal niche. The dual function that we describe here provides a physiological example of combined cell-autonomous and cell-nonautonomous functions of p21 with implications in self renewal, linking the relative quiescence of adult stem cells to their longevity and potentiality.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação da Expressão Gênica/genética , Células-Tronco Neurais/fisiologia , Fatores Etários , Animais , Bromodesoxiuridina , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ciclo Celular/genética , Diferenciação Celular/genética , Linhagem Celular Transformada , Meios de Cultivo Condicionados/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/deficiência , Antígeno Ki-67/metabolismo , Camundongos , Camundongos Knockout , Mutagênese , Células-Tronco Neoplásicas , Proteínas do Tecido Nervoso/metabolismo , Frações Subcelulares/metabolismo , Fatores de Tempo , Transdução Genética , TransfecçãoRESUMO
In the adult brain, continual neurogenesis of olfactory neurons is sustained by the existence of neural stem cells (NSCs) in the subependymal niche. Elimination of the cyclin-dependent kinase inhibitor 1A (p21) leads to premature exhaustion of the subependymal NSC pool, suggesting a relationship between cell cycle control and long-term self-renewal, but the molecular mechanisms underlying NSC maintenance by p21 remain unexplored. Here we identify a function of p21 in the direct regulation of the expression of pluripotency factor Sox2, a key regulator of the specification and maintenance of neural progenitors. We observe that p21 directly binds a Sox2 enhancer and negatively regulates Sox2 expression in NSCs. Augmented levels of Sox2 in p21 null cells induce replicative stress and a DNA damage response that leads to cell growth arrest mediated by increased levels of p19(Arf) and p53. Our results show a regulation of NSC expansion driven by a p21/Sox2/p53 axis.
Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , Inibidor de Quinase Dependente de Ciclina p21/genética , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Mutantes , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genéticaRESUMO
Stem cell division can result in two sibling cells exhibiting differential mitogenic and self-renewing potential. Here, we present evidence that the dual-specificity kinase Dyrk1A is part of a molecular pathway involved in the regulation of biased epidermal growth factor receptor (EGFR) signaling in the progeny of dividing neural stem cells (NSC) of the adult subependymal zone (SEZ). We show that EGFR asymmetry requires regulated sorting and that a normal Dyrk1a dosage is required to sustain EGFR in the two daughters of a symmetrically dividing progenitor. Dyrk1A is symmetrically or asymmetrically distributed during mitosis, and biochemical analyses indicate that it prevents endocytosis-mediated degradation of EGFR by a mechanism that requires phosphorylation of the EGFR signaling modulator Sprouty2. Finally, Dyrk1a heterozygous NSCs exhibit defects in self-renewal, EGF-dependent cell-fate decisions, and long-term persistence in vivo, suggesting that symmetrical divisions play a role in the maintenance of the SEZ reservoir.