RESUMO
Several mosaic mutations of the mammalian/mechanistic target of rapamycin (mTOR) have recently been found in patients with cortical malformations, such as hemimegalencephaly (HME) and focal cortical dysplasia (FCD). Although all of them should activate mTOR signaling, comparisons of the impact of different mTOR mutations on brain development have been lacking. Also it remains unknown if any potential differences these mutations may have on cortical development are directly related to a degree of mTOR signaling increase. The present study assessed levels of mTORC1 pathway activity in cell lines and rat primary neurons overexpressing several mTOR mutants that were previously found in HME, FCD, cancer patients and in vitro mutagenesis screens. Next we introduced the mutants, enhancing mTORC1 signaling most potently, into developing mouse brains and assessed electroporated cell morphology and migratory phenotype using immunofluorescent staining. We observed the differential inhibition of neuronal progenitor cortical migration, which partly corresponded with a degree of mTORC1 signaling enhancement these mutants induced in cultured cells. The most potent quadruple mutant prevented most of the progenitors from entering the cortical plate. Cells that expressed less potent, single-point, mTOR mutants entered the cortical plate but failed to reach its upper layers and had enlarged soma. Our findings suggest a correlation between the potency of mTOR mutation to activate mTORC1 pathway and disruption of cortical migration.
Assuntos
Córtex Cerebelar/embriologia , Mutação , Neurônios/citologia , Neurônios/enzimologia , Serina-Treonina Quinases TOR/genética , Animais , Movimento Celular/genética , Córtex Cerebelar/citologia , Córtex Cerebelar/enzimologia , Córtex Cerebelar/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/enzimologia , Embrião de Mamíferos/metabolismo , Células HEK293 , Humanos , Malformações do Desenvolvimento Cortical/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Neurogênese/genética , Neurônios/metabolismo , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
Neuronal-glial relationships play a critical role in the maintenance of central nervous system architecture and neuronal specification. A deeper understanding of these relationships can elucidate cellular cross-talk capable of sustaining proper development of neural tissues. In the cerebellum, cerebellar granule neuron precursors (CGNPs) proliferate in response to Purkinje neuron-derived Sonic hedgehog (Shh) before ultimately exiting the cell cycle and migrating radially along Bergmann glial fibers. However, the function of Bergmann glia in CGNP proliferation remains not well defined. Interestingly, the Hh pathway is also activated in Bergmann glia, but the role of Shh signaling in these cells is unknown. In this study, we show that specific ablation of Shh signaling using the tamoxifen-inducible TNCYFP-CreER line to eliminate Shh pathway activator Smoothened in Bergmann glia is sufficient to cause severe cerebellar hypoplasia and a significant reduction in CGNP proliferation. TNCYFP-CreER; SmoF/- (SmoCKO) mice demonstrate an obvious reduction in cerebellar size within two days of ablation of Shh signaling. Mutant cerebella have severely reduced proliferation and increased differentiation of CGNPs due to a significant decrease in Shh activity and concomitant activation of Wnt signaling in SmoCKO CGNPs, suggesting that this pathway is involved in cross-talk with the Shh pathway in regulating CGNP proliferation. In addition, Purkinje cells are ectopically located, their dendrites stunted, and the Bergmann glial network disorganized. Collectively, these data demonstrate a previously unappreciated role for Bergmann glial Shh signaling activity in the proliferation of CGNPs and proper maintenance of cerebellar architecture.
Assuntos
Córtex Cerebelar/embriologia , Proteínas Hedgehog/fisiologia , Neuroglia/fisiologia , Animais , Astrócitos/metabolismo , Diferenciação Celular , Divisão Celular , Proliferação de Células/fisiologia , Células Cultivadas , Córtex Cerebelar/fisiologia , Neoplasias Cerebelares/metabolismo , Cerebelo/anormalidades , Cerebelo/embriologia , Deficiências do Desenvolvimento/genética , Proteínas Hedgehog/metabolismo , Camundongos , Malformações do Sistema Nervoso/embriologia , Malformações do Sistema Nervoso/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Células de Purkinje/metabolismo , Transdução de Sinais , Via de Sinalização Wnt/genéticaRESUMO
Lead (Pb), a known environmental toxicant, adversely affects almost all organ systems. In this study, we investigated the effects of maternal lead exposure on fetal rat cerebellum. Female Sprague-Dawley rats were given lead nitrate in drinking water (0, 0.5, and 1%) for two weeks before conception, and during pregnancy. Fetuses were collected by caesarian section on gestational day 21 and observed for developmental abnormalities. The fetal cerebellar sections from control and 1% lead group were stained with cresyl violet. Immunohistochemical expressions of p53, Bax, Bcl-2, and caspase 3 were quantified by AnalySIS image analyzer (Life Science, Germany). Lead exposure induced developmental abnormalities of eyes, ear, limbs, neck and ventral abdominal wall; however, these abnormalities were commonly seen in the 1% lead-treated group. In addition, lead also caused fetal mortality and reduced body growth in both dose groups and reduced brain weight in the 1% lead-treated group. The fetal cerebella from the 1% lead-treated group showed unorganized cerebellar cortical layers, and degenerative changes in granule and Purkinje cells such as the formation of clumps of Nissl granules. An increase in Bax and caspase 3, and a decrease in Bcl-2 (p < 0.05), but not in p53, showed apoptosis of the neurons. In conclusion, gestational lead exposure in rats induces fetal toxicity and developmental abnormalities. The lead exposure also impairs development of cerebellar layers, induces structural changes, and apoptosis in the fetal cerebellar cortex. These results suggest that lead exposure during gestation is extremely toxic to developing cerebellum in rats.
Assuntos
Apoptose/efeitos dos fármacos , Córtex Cerebelar/efeitos dos fármacos , Anormalidades Congênitas/etiologia , Poluentes Ambientais/toxicidade , Chumbo/toxicidade , Exposição Materna/efeitos adversos , Nitratos/toxicidade , Organogênese/efeitos dos fármacos , Animais , Córtex Cerebelar/embriologia , Córtex Cerebelar/metabolismo , Córtex Cerebelar/patologia , Anormalidades Congênitas/metabolismo , Anormalidades Congênitas/patologia , Feminino , Imuno-Histoquímica , Gravidez , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Ratos Sprague-Dawley , Ratos Wistar , Proteína Supressora de Tumor p53/biossíntese , Proteína X Associada a bcl-2/biossínteseRESUMO
The present study was aimed at supplementing our previous investigations on the morphological features of the Purkinje cells during the autonomic nervous system development, particularly in victims of sudden perinatal death (Sudden Intrauterine Unexplained Death Syndrome and Sudden Infant Death Syndrome), given their crucial role in determining connectivity patterns in the brain as well as in the control of autonomic functions. We highlighted in these pathologies, and precisely in 21 cases of sudden foetal death and 26 cases of sudden infant death, a high percentage of developmental defects of the Purkinje cells such as heterotopia, hypoplasia, hyperplasia, mitotic and/or shrunken features and abnormal neuronal nuclear antigen expression. These alterations can be interpreted as a result of a defective maturation and/or migration of Purkinje cells in foetal cerebellum, likely consequence of exposure to injuries, particularly to maternal cigarette smoke. Interestingly, we observed in sudden perinatal deaths an association with similar developmental defects of both the dentate and the inferior olivary nuclei. This suggests the existence of a Purkinje-Olivo-Dentate network playing a fundamental role in triggering a sudden death mechanism in perinatal life in the presence of specific risk factors.
Assuntos
Morte Fetal/diagnóstico , Efeitos Tardios da Exposição Pré-Natal/diagnóstico , Células de Purkinje/patologia , Fumar/efeitos adversos , Fumar/patologia , Morte Súbita do Lactente/diagnóstico , Córtex Cerebelar/efeitos dos fármacos , Córtex Cerebelar/embriologia , Córtex Cerebelar/patologia , Feminino , Morte Fetal/epidemiologia , Humanos , Lactente , Recém-Nascido , Nicotina/administração & dosagem , Nicotina/toxicidade , Gravidez , Efeitos Tardios da Exposição Pré-Natal/epidemiologia , Células de Purkinje/efeitos dos fármacos , Morte Súbita do Lactente/epidemiologiaRESUMO
Asymmetric cell divisions are a fundamental feature of neural development, and misregulation can lead to brain abnormalities or tumor formation. During an asymmetric cell division, molecular determinants are segregated preferentially into one daughter cell to specify its fate. An important goal is to identify the asymmetric determinants in neural progenitor cells, which could be tumor suppressors or inducers of specific neural fates. Here, we show that the double-stranded RNA-binding protein Stau2 is distributed asymmetrically during progenitor divisions in the developing mouse cortex, preferentially segregating into the Tbr2(+) neuroblast daughter, taking with it a subset of RNAs. Knockdown of Stau2 stimulates differentiation and overexpression produces periventricular neuronal masses, demonstrating its functional importance for normal cortical development. We immunoprecipitated Stau2 to examine its cargo mRNAs, and found enrichment for known asymmetric and basal cell determinants, such as Trim32, and identified candidates, including a subset involved in primary cilium function.
Assuntos
Divisão Celular Assimétrica , Córtex Cerebelar/embriologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Células Cultivadas , Córtex Cerebelar/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Knockout , Análise em Microsséries , Proteínas do Tecido Nervoso/genética , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Purkinje cells are the sole output neurons of the cerebellar cortex and their dysfunction causes severe ataxia. We found that Purkinje cells could be robustly generated from mouse embryonic stem (ES) cells by recapitulating the self-inductive signaling microenvironments of the isthmic organizer. The cell-surface marker Neph3 enabled us to carry out timed prospective selection of Purkinje cell progenitors, which generated morphologically characteristic neurons with highly arborized dendrites that expressed mature Purkinje cell-specific markers such as the glutamate receptor subunit GluRδ2. Similar to mature Purkinje cells, these neurons also showed characteristic spontaneous and repeated action potentials and their postsynaptic excitatory potentials were generated exclusively through nonNMDA glutamate receptors. Fetal transplantation of precursors isolated by fluorescence-activated cell sorting showed orthotopic integration of the grafted neurons into the Purkinje cell layer with their axons extending to the deep cerebellar nuclei and dendrites receiving climbing and parallel fibers. This selective preparation of bona fide Purkinje cells should aid future investigation of this important neuron.
Assuntos
Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células de Purkinje/fisiologia , Células-Tronco/fisiologia , Animais , Células Cultivadas , Córtex Cerebelar/citologia , Córtex Cerebelar/embriologia , Embrião de Mamíferos , Feminino , Citometria de Fluxo/métodos , Regulação da Expressão Gênica no Desenvolvimento/genética , Glutamato Descarboxilase/genética , Proteínas de Fluorescência Verde/genética , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Patch-Clamp/métodos , GravidezRESUMO
The aims of this study were to investigate in the human cerebellar cortex the structural and biological ontogenetic features, the possible presence of alterations in cases of sudden unexplained fetal and infant death, and the involvement of the maternal cigarette smoking in developmental abnormalities. We analyzed 52 brains of fetal and infant death victims, aged from the second gestational trimester to 12th postnatal month. In the cerebellar cortex we evaluated, besides the morphological aspects, the expression of several biomarkers implicated in proliferative processes (c-fos, proliferating cell nuclear antigen, and apoptosis) as well as the presence of the neurotransmitter somatostatin, which is strongly implicated in central nervous system differentiation, and of EN2 gene. The observed features of the cerebellar cortex, mainly confined to the transient external granular layer, were high proliferative activity and high expression of both somatostatin and EN2 gene in prenatal life and high apoptotic index after birth. In 41% of the sudden unexplained death victims, in the greater part with smoking mothers, we observed different biopathological alterations of the cerebellar cortex. Maternal smoking is increasingly being demonstrated to be one of the main contributors to developmental neurological alterations in the offspring.
Assuntos
Córtex Cerebelar/embriologia , Córtex Cerebelar/patologia , Morte Súbita do Lactente/patologia , Apoptose , Proteínas de Homeodomínio/análise , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Proteínas do Tecido Nervoso/análise , Antígeno Nuclear de Célula em Proliferação/análise , Proteínas Proto-Oncogênicas c-fos/análise , Somatostatina/análiseRESUMO
The plasma membrane Ca(2+)-ATPase (PMCA) is highly expressed in the nervous system, but little information is available about its implication in neuronal development. We have analyzed the expression and localization of different isoforms of PMCA in membrane vesicles and sections of chick cerebellum from embryonic day 10 to hatching. We found that the relative amount of each PMCA isoform and their spatiotemporal distribution in the cerebellum are directly linked to precise cellular types during the cerebellar maturation, even in a non-neural tissue as choroid plexus. Purkinje cells contain the highest diversity of PMCA isoforms of the cerebellar cortex since the moment of its morphogenesis. From embryonic day 15, the PMCA2 was highly expressed in the whole Purkinje cell, while PMCAs 1 and 3 had a more restricted distribution in the soma and dendritic branches, and these distributions were evolving according with cell maturation. Other cellular types seem to contain a specific combination of isoforms, but with a well-defined distribution pattern at late moments of development. Thus, PMCAs 1 and 3 were located in the soma of molecular layer interneurons, and only the PMCA2 was observed in granule cells at hatching. Furthermore, PMCA isoforms are also expressed in cellular compartments characterized by a high amount of synapses, suggesting a key role of these proteins in synaptogenesis and in the maturation of neuronal electrophysiological properties.
Assuntos
Cerebelo/embriologia , Cerebelo/enzimologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Animais Recém-Nascidos , Membrana Celular/enzimologia , Córtex Cerebelar/embriologia , Córtex Cerebelar/enzimologia , Embrião de Galinha , Galinhas , Plexo Corióideo/embriologia , Plexo Corióideo/enzimologia , Isoenzimas/metabolismo , Células de Purkinje/enzimologia , Sinapses/enzimologia , Distribuição TecidualRESUMO
Medulloblastoma, the most common brain tumor in childhood, appears to originate from cerebellar granule cell precursors (GCPs), located in the external granular layer (EGL) of the cerebellum. The antiproliferative gene PC3 (Tis21/BTG2) promotes cerebellar neurogenesis by inducing GCPs to shift from proliferation to differentiation. To assess whether PC3 can prevent the neoplastic transformation of GCPs and medulloblastoma development, we crossed transgenic mice conditionally expressing PC3 (TgPC3) in GCPs with Patched1 heterozygous mice (Ptc(+/-)), a model of medulloblastoma pathogenesis characterized by hyperactivation of the Sonic Hedgehog pathway. Perinatal up-regulation of PC3 in Ptc(+/-)/TgPC3 mice results in a decrease of medulloblastoma incidence of approximately 40% and in a marked reduction of preneoplastic abnormalities, such as hyperplastic EGL areas and lesions. Moreover, overexpression of cyclin D1, hyperproliferation, and defective differentiation--observed in Ptc(+/-) GCPs--are restored to normality in Ptc(+/-)/TgPC3 mice. The PC3-mediated inhibition of cyclin D1 expression correlates with recruitment of PC3 to the cyclin D1 promoter, which is accompanied by histone deacetylation. Remarkably, down-regulation of PC3 is observed in preneoplastic lesions, as well as in human and murine medulloblastomas. As a whole, this indicates that PC3 may prevent medulloblastoma development by controlling cell cycle and promoting differentiation of GCPs.
Assuntos
Neoplasias Cerebelares/prevenção & controle , Genes Supressores de Tumor , Proteínas Imediatamente Precoces/fisiologia , Meduloblastoma/prevenção & controle , Acetilação , Animais , Síndrome do Nevo Basocelular/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Divisão Celular/genética , Divisão Celular/fisiologia , Transformação Celular Neoplásica/genética , Córtex Cerebelar/anormalidades , Córtex Cerebelar/embriologia , Neoplasias Cerebelares/genética , Coristoma/genética , Imunoprecipitação da Cromatina , Ciclina D , Ciclinas/biossíntese , Ciclinas/genética , Proteínas Hedgehog/fisiologia , Heterozigoto , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Meduloblastoma/genética , Camundongos , Camundongos Transgênicos , Síndromes Neoplásicas Hereditárias/genética , Síndromes Neoplásicas Hereditárias/prevenção & controle , Neurônios/patologia , Células PC12/química , Receptores Patched , Receptor Patched-1 , Lesões Pré-Cancerosas/genética , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , RNA Neoplásico/genética , Ratos , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Supressoras de TumorRESUMO
Probing undiscovered neurosubstances that play important roles in the regulation of cerebellar function is essential for the progress of our understanding of the cerebellum. New findings over the past decade have established that the cerebellum as well as other brain regions synthesizes steroids de novo from cholesterol through mechanisms at least partly independent of peripheral steroidogenic glands. Such steroids synthesized de novo in the brain are called neurosteroids. Recently the Purkinje cell, a cerebellar neuron, has been identified as a major site for neurosteroid formation in the brain. This is the first demonstration of de novo neuronal neurosteroidogenesis in the brain. In mammals, the Purkinje cell actively synthesizes progesterone de novo from cholesterol during neonatal life, when cerebellar cortical formation occurs. 3alpha,5alpha-Tetrahydroprogesterone (allopregnanolone) is metabolized from progesterone in the neonatal cerebellum. Estrogen formation in the Purkinje cell may also occur in the neonate. Subsequently, recent studies on mammals using the Purkinje cell have demonstrated organizing actions of neurosteroids. Both progesterone and estradiol promote dendritic growth, spinogenesis and synaptogenesis via each cognate nuclear receptor in Purkinje neurons. Allopregnanolone is also involved in Purkinje and granule cell survival. Thus the Purkinje cell serves as an excellent cellular model for understanding the formation of cerebellar neuronal circuit in relation to organizing actions of neurosteroids.
Assuntos
Diferenciação Celular/fisiologia , Córtex Cerebelar/embriologia , Córtex Cerebelar/crescimento & desenvolvimento , Células de Purkinje/metabolismo , Esteroides/biossíntese , Animais , Córtex Cerebelar/metabolismo , Estrogênios/biossíntese , Humanos , Vias Neurais/embriologia , Vias Neurais/crescimento & desenvolvimento , Vias Neurais/metabolismo , Pregnanolona/biossíntese , Progesterona/biossíntese , Células de Purkinje/citologia , Receptores de Esteroides/metabolismoRESUMO
In vertebrates, the receptor families deleted in colorectal cancer (DCC) and UNC5 mediate responses to the bifunctional guidance cue netrin-1. DCC mediates attraction, whereas a complex of DCC and UNC5 mediates repulsion. Thus, a primary determinant of the responsiveness of an axon to netrin-1 is the presence or absence of UNC5 family members on the cell surface. Currently, little is known about the role of receptor trafficking in regulating neuronal responses to netrin-1. We show that protein interacting with C-kinase 1 (PICK1) recruits activated protein kinase Calpha (PKCalpha) to MycUNC5A at the plasma membrane, stimulating its endocytosis. We identify two PKCalpha phosphorylation sites at serines 408 and 587, as well as dileucine internalization motifs, which are required for this endocytosis. We find that PKCalpha-stimulated internalization of UNC5A alters the functional response of developing hippocampal axons to netrin-1, preventing UNC5A-mediated growth cone collapse and converting netrin-1-stimulated chemorepulsion to attraction. To address whether this conversion in axonal response occurs in neurons expressing endogenous levels of UNC5, we show that mouse cerebellar granule axons exhibit chemorepulsion in a netrin-1 gradient and that this chemorepulsion is converted to chemoattraction after PKCalpha activation. We demonstrate that this repulsion depends on UNC5A because Unc5a-/- axons are not repelled and show this conversion depends on PICK1 because PICK1-/- axons are not converted to chemoattraction after PKCalpha activation. Together, these data provide a potential mechanism to explain how developing neurons alter their responsiveness to netrin-1 at intermediate choice points as they navigate to their targets.
Assuntos
Proteínas de Transporte/metabolismo , Sistema Nervoso Central/embriologia , Cones de Crescimento/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas Nucleares/metabolismo , Proteína Quinase C-alfa/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Comunicação Celular/fisiologia , Membrana Celular/metabolismo , Células Cultivadas , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Córtex Cerebelar/citologia , Córtex Cerebelar/embriologia , Córtex Cerebelar/metabolismo , Fatores Quimiotáticos/metabolismo , Quimiotaxia/fisiologia , Sinais (Psicologia) , Proteínas do Citoesqueleto , Endocitose/fisiologia , Ativação Enzimática/fisiologia , Cones de Crescimento/ultraestrutura , Hipocampo/citologia , Hipocampo/embriologia , Hipocampo/metabolismo , Camundongos , Camundongos Knockout , Receptores de Netrina , Netrina-1 , Fosforilação , Ratos , Receptores de Superfície Celular/metabolismoRESUMO
Astrocyte death has been implicated in several neuropathological diseases, but the identification of molecules susceptible of promoting astrocyte survival has been elusive. We investigated whether transforming growth factor alpha (TGFalpha), an erbB1/EGFR ligand, which promotes glioma progression and affects astrocyte metabolism at embryonic and adult stages, regulates astrocyte survival. Primary serum-free astrocyte cultures from post-natal mouse and fetal human cortices were used. Transforming growth factor alpha protected both species of astrocytes from staurosporine-induced apoptosis. In serum-free medium, mouse astrocytes did not survive beyond 2 months while TGFalpha-treated astrocytes survived up to 12 months. Transforming growth factor alpha also promoted long-term survival of human astrocytes. We additionally extended TGFalpha proliferative effects to human astrocytes. After 3 days of permanent application, TGFalpha induced a major downregulation of both erbB1 and erbB2. This downregulation did not impair the functional activation of the receptors, as ascertained by their tyrosine phosphorylation and the continuous stimulation of both ERK/MAPK and PI3K/Akt pathways up to 7 days, the longest time examined. The full cellular effects of TGFalpha required activation of both transduction pathways. Enhanced proliferation and survival thus define TGFalpha as a gliatrophin for mammalian astrocytes. These results demonstrate that in normal, non-transformed astrocytes, sustained and functional erbBs activation is achieved without bypassing ligand-induced receptors downregulation.
Assuntos
Astrócitos/metabolismo , Regulação para Baixo/efeitos dos fármacos , Receptores ErbB/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptor ErbB-2/metabolismo , Fator de Crescimento Transformador alfa/farmacologia , Envelhecimento/metabolismo , Animais , Astrócitos/citologia , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebelar/citologia , Córtex Cerebelar/embriologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glioma/metabolismo , Humanos , Camundongos , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador alfa/metabolismoRESUMO
Because it is now possible to obtain high-resolution multiplanar MR imaging of the cerebellum and because of the developing interest on the role of the cerebellum on higher brain functions, we have decided to study the process of cerebellar fissuration. All brain MRI examinations performed in children for varied neurological and neurosurgical indications, especially children with non-specific mental retardation and patients with cerebral malformation detected at initial imaging work-up, were reviewed. Fissuration and lobulation anomalies (abnormal orientation of fissures, pseudopolymicrogyria, cortical thickening, subcortical cysts and heterotopia) were identified that we called cerebellar cortical dysplasia (CCD). In order to better understand the origin of this malformation, current data on cerebellar embryogenesis and histogenesis will be reviewed, and the pathological and radiological features will be illustrated. Milder forms of CCD represent a distinct group of anomalies that should be distinguished from other types of cerebellar dysplasia (agenesis, hypoplasia or complex dysplasia with involvement of the cerebellar vermis (rhombencephalosynapsis)) or combined cerebellar and cerebral dysplasia (muscular dystrophies and lissencephaly). Recognition of cerebellar cortical dysplasia could be a first step towards a broader understanding of its pathogenesis and significance.
Assuntos
Córtex Cerebelar/anormalidades , Córtex Cerebelar/patologia , Doenças Cerebelares/diagnóstico , Imageamento por Ressonância Magnética , Adolescente , Ataxia/etiologia , Córtex Cerebelar/embriologia , Doenças Cerebelares/complicações , Doenças Cerebelares/embriologia , Criança , Pré-Escolar , Deficiências do Desenvolvimento/etiologia , Diagnóstico Diferencial , Epilepsia/etiologia , Feminino , Humanos , Lactente , Recém-Nascido , Deficiência Intelectual/etiologia , Imageamento por Ressonância Magnética/métodos , Imageamento por Ressonância Magnética/normas , Masculino , Seleção de Pacientes , Estudos Prospectivos , Reprodutibilidade dos Testes , Estudos Retrospectivos , Fatores de Risco , Índice de Gravidade de DoençaRESUMO
Recent studies have shown that the mammalian cerebellar cortex can be subdivided into a reproducible array of zones and stripes. In particular, discontinuous patterns of gene expression together with mutational analysis suggest that there are at least four distinct transverse zones along the rostrocaudal axis in mouse: the anterior zone (lobules I-V), the central zone (lobules VI and VII), the posterior zone (lobules VIII and IX), and the nodular zone (lobule X). Here we show that the divergent homeobox-containing transcription factor, Tlx- 3 (also known as Hox11L2 or Rnx) is transiently expressed in external granule cells in a distinct transverse domain of the developing chick cerebellar cortex. Expression is first detected at Hamburger and Hamilton (HH) stage 35. Interestingly, Tlx-3 mRNA expression is initially confined to, and coincident with, the morphological development of fissures. Slightly later, at HH stage 38, expression extends throughout the developing external granular layer (EGL) of lobules I-IXab. Notably, no Tlx-3 expression was detected in lobules IXc and X at any developmental time point examined. Expression is noticeably stronger in nonproliferating cells located in the deep layer of the EGL. Tlx-3 expression is downregulated as granule cells migrate inward to form the internal granule layer and is undetectable shortly after birth. These results suggest that Tlx-3 is expressed as granule cells become postmitotic and suggest that Tlx-3 may play a role in the differentiation of distinct neuronal populations in the cerebellum.
Assuntos
Padronização Corporal/genética , Diferenciação Celular/genética , Córtex Cerebelar/embriologia , Córtex Cerebelar/metabolismo , Embrião de Galinha/embriologia , Embrião de Galinha/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/genética , Proteínas Oncogênicas/genética , Animais , Córtex Cerebelar/citologia , Embrião de Galinha/citologia , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Mitose/fisiologia , Neurônios/citologia , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição SOXC , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
A previously uncharacterized 4.5-kb mouse cDNA clone, designated mc7, was isolated and found to be predominantly expressed in brain. This cDNA predicts a 1035-bp open reading frame that encodes for a 345-amino acid polypeptide especially rich in glutamic acid residues located in the region from residues 80 to 174. Computational analysis revealed among other features, putative zinc-finger motifs and coiled-coil regions. The corresponding mc7 gene is detected in mouse, rat, pig and human genomes. In mouse the mc7 mRNA is expressed predominantly in brain and to a much lesser extent in kidney, lung and spleen. In brain it is detectable as early as embryonic day 14 while it is retained in the adult. In situ hybridization studies revealed that mc7 mRNA is widely, albeit unevenly, expressed in neurons throughout the adult brain. Developmental in situ hybridization studies in the cerebellar cortex demonstrated that at postnatal day 5 mc7 mRNA is mainly expressed in neuroblasts of the external granular layer and in developing neurons of the internal granular layer. Some staining is also present in purkinje cells becoming particularly pronounced at postnatal day 10, the time of arborarization of their dendritic tree. In the adult cerebellar cortex expression is mainly confined in purkinje cells and to a lesser extent in granule neurons. The early expression of mc7 in neuroblasts and developing neurons as well as its retention in a wide variety of mature neurons suggest that it may play a role in the process of differentiation and maturation of these cells in the brain.
Assuntos
Química Encefálica/genética , Proteínas de Transporte/genética , Córtex Cerebelar/citologia , Proteínas do Tecido Nervoso/genética , Neurônios/fisiologia , Células-Tronco/fisiologia , Fatores Etários , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Córtex Cerebelar/embriologia , Clonagem Molecular , DNA Complementar/isolamento & purificação , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Dados de Sequência Molecular , Neurônios/citologia , RNA Mensageiro/análise , Células-Tronco/citologia , Proteínas com Motivo Tripartido , Dedos de Zinco/genéticaRESUMO
The formation of covalent isopeptide cross-links between cell surface protein molecules by the enzyme transglutaminase C influences cell adhesion and morphology. Retinoid-inducible cross-linking activity associated with this enzyme is present in the developing rat cerebellar cortex [Perry M. J. M. et al. (1995) Neuroscience 65, 1063-1076]. A monoclonal antibody was used to localize transglutaminase C to granule neurons in the developing cerebellar cortex. The enzyme was inducible by retinoic acid both in granule neurons cultured from postnatal rat cerebellar cortex and in cells of the embryonic dorsal rhombic lip, which contain granule neuron precursors. A possible biological function for transglutaminase activity was investigated in living granule neurons, cultured on a biomatrix substratum, studied by time-lapse cinematographic analysis using the transglutaminase inactivator RS-48373-007. Inhibition of cross-linking activity did not influence the number of neurites formed by granule neurons, but caused the destabilization of neurites during the initial outgrowth period, seen as an increase in the number of growth cone retractions and the onset of premature axon collateral formation (bifurcation). Inactivation of cross-linking activity prevented the formation of fascicles between neurites only when cells were cultured on a biomatrix surface. Two glial proteins involved in cell-extracellular matrix interactions, midkine and galectin-3, were identified as putative substrates for granule neuron transglutaminase. The results suggest that covalent cross-link formation by transglutaminase C or a related enzyme generates multimeric molecular forms of glial-derived proteins, and plays a role in stabilizing newly formed neurites. A possible non-pathological role for transglutaminase in the control of axon collateral branching by developing granule neurons in the cerebellar cortex is discussed.
Assuntos
Antígenos de Diferenciação/metabolismo , Proteínas de Transporte/metabolismo , Diferenciação Celular/efeitos dos fármacos , Córtex Cerebelar/embriologia , Córtex Cerebelar/crescimento & desenvolvimento , Citocinas , Proteínas de Ligação ao GTP/metabolismo , Neuritos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Transglutaminases/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Diferenciação Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Córtex Cerebelar/citologia , Córtex Cerebelar/enzimologia , Reagentes de Ligações Cruzadas/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Feminino , Galectina 3 , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/enzimologia , Cones de Crescimento/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Midkina , Neuritos/enzimologia , Neuritos/ultraestrutura , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neuroglia/enzimologia , Neurônios/citologia , Neurônios/enzimologia , Gravidez , Proteína 2 Glutamina gama-Glutamiltransferase , Tretinoína/metabolismo , Tretinoína/farmacologiaAssuntos
Axônios/fisiologia , Córtex Cerebelar/citologia , Cones de Crescimento/fisiologia , Fibras Nervosas/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas de Peixe-Zebra , Agrina/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Diferenciação Celular , Córtex Cerebelar/embriologia , Quinase 3 da Glicogênio Sintase , Junção Neuromuscular/ultraestrutura , Plasticidade Neuronal , Fosforilação , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Sinapses/fisiologia , Proteínas WntRESUMO
Synapse formation requires changes in cell morphology and the upregulation and localization of synaptic proteins. In the cerebellum, mossy fibers undergo extensive remodeling as they contact several granule cells and form complex, multisynaptic glomerular rosettes. Here we show that granule cells secrete factors that induce axon and growth cone remodeling in mossy fibers. This effect is blocked by the WNT antagonist, sFRP-1, and mimicked by WNT-7a, which is expressed by granule cells. WNT-7a also induces synapsin I clustering at remodeled areas of mossy fibers, a preliminary step in synaptogenesis. Wnt-7a mutant mice show a delay in the morphological maturation of glomerular rosettes and in the accumulation of synapsin I. We propose that WNT-7a can function as a synaptogenic factor.
Assuntos
Axônios/fisiologia , Córtex Cerebelar/citologia , Glicoproteínas , Cones de Crescimento/fisiologia , Fibras Nervosas/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Animais , Axônios/ultraestrutura , Fatores Biológicos/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Diferenciação Celular , Células Cultivadas , Córtex Cerebelar/embriologia , Técnicas de Cocultura , Meios de Cultivo Condicionados , Meios de Cultura Livres de Soro/farmacologia , Dendritos/fisiologia , Dendritos/ultraestrutura , Quinase 3 da Glicogênio Sintase , Inositol/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Lítio/farmacologia , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Plasticidade Neuronal , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais , Sinapses/fisiologia , Sinapsinas/metabolismo , Transfecção , Proteínas WntRESUMO
In the developing chicken cerebellar cortex, three cadherins (Cad6B, Cad7, and R-cadherin) are expressed in distinct parasagittal segments that are separated from each other by ribbons of migrating interneurons and granule cells which express R-cadherin and Cad7, respectively. The segment/ribbon pattern is respected by the expression of other types of molecules, such as engrailed-2 and SC1/BEN/DM-GRASP. The cadherin-defined segments contain young Purkinje cells which are connected to underlying nuclear zones expressing the same cadherin, thereby forming parasagittal cortico-nuclear zones of topographically organized connections. In addition, R-cadherin-positive mossy fiber terminals display a periodic pattern in the internal granular layer. In this layer, Cad7 and R-cadherin are associated with synaptic complexes. These results suggest that cadherins play a pivotal role in the formation of functional cerebellar architecture by providing a three-dimensional scaffold of adhesive information.
Assuntos
Caderinas/biossíntese , Córtex Cerebelar/embriologia , Animais , Antígenos de Diferenciação , Proteínas Aviárias , Mapeamento Encefálico , Córtex Cerebelar/metabolismo , Núcleos Cerebelares/embriologia , Núcleos Cerebelares/metabolismo , Embrião de Galinha , Sinapses/metabolismoRESUMO
In the adult cerebellar cortex, the differential expression of neuronal nitric oxide synthase (nNOS) can be used to discern discrete subsets of granule neurons. These subsets originate concurrently with the afferent innervation of granule neurons. Using primary cultures established from murine cerebella at different development stages, we analyzed the effects of the neurotransmitter GABA and the neurotrophins BDNF and NT-3 on nNOS expression in developing granule cells. We show a biophasic effect of GABA on nNOS expression, which correlates with the ability of this transmitter to increase intracellular calcium levels. BDNF and NT-3 are shown to increase nNOS levels in cultures derived from postnatal, but not in those from embryonic cerebella. Together with previous findings [Baader and Schilling; J Neurosci 1996; 16:1440-1449], these data suggest a scenario in which afferent innervation and growth factors cooperate to differentially affect nNOS expression at discrete developmental stages of cerebellar granule cells.