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1.
Int J Mol Sci ; 24(16)2023 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-37629145

RESUMO

The apical dendrite of a cortical projection neuron (CPN) is generated from the leading process of the migrating neuron as the neuron completes migration. This transformation occurs in the cortical marginal zone (MZ), a layer that contains the Cajal-Retzius neurons and their axonal projections. Cajal-Retzius neurons (CRNs) are well known for their critical role in secreting Reelin, a glycoprotein that controls dendritogenesis and cell positioning in many regions of the developing brain. In this study, we examine the possibility that CRNs in the MZ may provide additional signals to arriving CPNs, that may promote the maturation of CPNs and thus shape the development of the cortex. We use whole embryonic hemisphere explants and multiphoton microscopy to confirm that CRNs display intracellular calcium transients of <1-min duration and high amplitude during early corticogenesis. In contrast, developing CPNs do not show high-amplitude calcium transients, but instead show a steady increase in intracellular calcium that begins at the time of dendritic initiation, when the leading process of the migrating CPN is encountering the MZ. The possible existence of CRN to CPN communication was revealed by the application of veratridine, a sodium channel activator, which has been shown to preferentially stimulate more mature cells in the MZ at an early developmental time. Surprisingly, veratridine application also triggers large calcium transients in CPNs, which can be partially blocked by a cocktail of antagonists that block glutamate and glycine receptor activation. These findings outline a model in which CRN spontaneous activity triggers the release of glutamate and glycine, neurotransmitters that can trigger intracellular calcium elevations in CPNs. These elevations begin as CPNs initiate dendritogenesis and continue as waves in the post-migratory cells. Moreover, we show that the pharmacological blockade of glutamatergic signaling disrupts migration, while forced expression of a bacterial voltage-gated calcium channel (CavMr) in the migrating neurons promotes dendritic growth and migration arrest. The identification of CRN to CPN signaling during early development provides insight into the observation that many autism-linked genes encode synaptic proteins that, paradoxically, are expressed in the developing cortex well before the appearance of synapses and the establishment of functional circuits.


Assuntos
Sinalização do Cálcio , Cálcio , Veratridina , Neurônios , Dendritos , Cálcio da Dieta , Ácido Glutâmico
2.
Front Neurosci ; 17: 1158419, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37250402

RESUMO

The adhesion systems employed by migrating cortical neurons are not well understood. Genetic deletion studies of focal adhesion kinase (FAK) and paxillin in mice suggested that these classical focal adhesion molecules control the morphology and speed of cortical neuron migration, but whether ß1 integrins also regulate migration morphology and speed is not known. We hypothesized that a ß1 integrin adhesion complex is required for proper neuronal migration and for proper cortical development. To test this, we have specifically deleted ß1 integrin from postmitotic migrating and differentiating neurons by crossing conditional ß1 integrin floxed mice into the NEX-Cre transgenic line. Similar to our prior findings with conditional paxillin deficiency, we found that both homozygous and heterozygous deletion of ß1 integrin causes transient mispositioning of cortical neurons in the developing cortex when analyzed pre- and perinatally. Paxillin and ß1 integrin colocalize in the migrating neurons and deletion of paxillin in the migrating neuron causes an overall reduction of the ß1 integrin immunofluorescence signal and reduction in the number of activated ß1 integrin puncta in the migrating neurons. These findings suggest that these molecules may form a functional complex in migrating neurons. Similarly, there was an overall reduced number of paxillin+ puncta in the ß1 integrin deficient neurons, despite the normal distribution of FAK and Cx26, a connexin required for cortical migration. The double knockout of paxillin and ß1 integrin produces a cortical malpositioning phenotype similar to the paxillin or ß1 integrin single knockouts, as would be expected if paxillin and ß1 integrin function on a common pathway. Importantly, an isolation-induced pup vocalization test showed that ß1 integrin mutants produced a significantly smaller number of calls compared to their littermate controls when analyzed at postnatal day 4 (P4) and revealed a several days trend in reduced vocalization development compared to controls. The current study establishes a role for ß1 integrin in cortical development and suggests that ß1 integrin deficiency leads to migration and neurodevelopmental delays.

3.
Transpl Infect Dis ; 24(1): e13772, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34905653

RESUMO

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with increased morbidity and mortality in solid organ transplant (SOT) recipients. Despite exclusion from SARS-CoV-2 vaccine clinical trials, these individuals were identified as high-risk and prioritized for vaccination in public health guidelines. METHODS: We prospectively evaluated humoral and cellular immune responses to two doses of the SARS-CoV-2 mRNA vaccine, BNT162b2, in 56 SOT recipients and 26 healthy controls (HCs). Blood specimens collected from participants prior to each dose and following the second dose were tested for SARS-CoV-2-specific antibodies, as well as CD4+ and CD8+ T-cell responses. RESULTS: SOT recipients demonstrated lower mean anti-SARS-CoV-2 antibody levels compared to HCs after each dose, and only 21.6% achieved an antibody response after the second dose within the range of HC responses. Similarly, the percentage of responsive CD4+ and CD8+ T cells in SOT recipients was lower than in HCs. While most HCs showed notable humoral and cellular responses, responses were less concordant in SOT recipients, with some showing evidence of either humoral or cellular response, but not both. CONCLUSION: Humoral and cellular immune responses to the BNT162b2 vaccine are markedly reduced in SOT recipients as compared to HCs, suggesting that SOT recipients may benefit from more tailored regimens such as higher dose and/or additional vaccinations.


Assuntos
COVID-19 , Transplante de Órgãos , Anticorpos Antivirais , Vacina BNT162 , Vacinas contra COVID-19 , Humanos , Imunidade Celular , SARS-CoV-2 , Transplantados , Vacinas Sintéticas , Vacinas de mRNA
4.
Mol Neurobiol ; 58(10): 5210-5223, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34272687

RESUMO

Fetal alcohol syndrome (FAS) is characterized by disrupted fetal brain development and postnatal cognitive impairment. The targets of alcohol are diverse, and it is not clear whether there are common underlying molecular mechanisms producing these disruptions. Prior work established that acute ethanol exposure causes a transient increase in tyrosine phosphorylation of multiple proteins in cultured embryonic cortical cells. In this study, we show that a similar tyrosine phosphorylation transient occurs in the fetal brain after maternal dosing with ethanol. Using phospho-specific antibodies and immunohistochemistry, we mapped regions of highest tyrosine phosphorylation in the fetal cerebral cortex and found that areas of dendritic and axonal growth showed elevated tyrosine phosphorylation 10 min after maternal ethanol exposure. These were also areas of Src expression and Src family kinase (SFK) activation loop phosphorylation (pY416) expression. Importantly, maternal pretreatment with the SFK inhibitor dasatinib completely prevents both the pY416 increase and the tyrosine phosphorylation response. The phosphorylation response was observed in the perisomatic region and neurites of immature migrating and differentiating primary neurons. Importantly, the initial phosphotyrosine transient (~ 30 min) targets both Src and Dab1, two critical elements in Reelin signaling, a pathway required for normal cortical development. This initial phosphorylation response is followed by sustained reduction in Ser3 phosphorylation of n-cofilin, a critical actin severing protein and an identified downstream effector of Reelin signaling. This biochemical disruption is associated with sustained reduction of F-actin content and disrupted Golgi apparatus morphology in developing cortical neurons. The finding outlines a model in which the initial activation of SFKs by ethanol has the potential to disrupt multiple developmentally important signaling systems for several hours after maternal exposure.


Assuntos
Córtex Cerebral/embriologia , Córtex Cerebral/enzimologia , Desenvolvimento Embrionário/efeitos dos fármacos , Etanol/toxicidade , Efeitos Tardios da Exposição Pré-Natal/enzimologia , Quinases da Família src/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Desenvolvimento Embrionário/fisiologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente
5.
eNeuro ; 7(4)2020.
Artigo em Inglês | MEDLINE | ID: mdl-32641498

RESUMO

Disruptions in neuronal dendrite development alter brain circuitry and are associated with debilitating neurological disorders. Nascent apical dendrites of cortical excitatory neurons project into the marginal zone (MZ), a cell-sparse layer characterized by intense chondroitin sulfate proteoglycan (CSPG) expression. Paradoxically, CSPGs are known to broadly inhibit neurite growth and regeneration. This raises the possibility that the growing apical dendrite is somehow insensitive to CSPG-mediated neurite growth inhibition. To test this, developing cortical neurons were challenged with both soluble CSPGs and CSPG-positive stripe substrates in vitro Soluble CSPGs inhibited dendritic growth and cortical dendrites respected CSPG stripe boundaries, effects that could be counteracted by prior CSPG inactivation by chondroitinase. Importantly, addition of Reelin, an extracellular signaling protein highly expressed in the MZ, partially rescued dendritic growth in the presence of CSPGs. High-resolution confocal imaging revealed that the CSPG-enriched areas of the MZ spatially correspond with the areas of reduced dendritic density in the Reelin null (reeler) cortex compared with controls. Chondroitinase injections into reeler explants resulted in increased dendritic growth into the MZ, recovering to near wild-type levels. Activation of the serine threonine kinase Akt is required for Reelin-dependent dendritic growth and we find that CSPGs induce Akt dephosphorylation, an effect that can be counteracted by Reelin addition. In contrast, CSPG application had no effect on the cytoplasmic adaptor Dab1, which is rapidly phosphorylated in response to Reelin and is upstream of Akt. These findings suggest CSPGs do inhibit cortical dendritic growth, but this effect can be counteracted by Reelin signaling.


Assuntos
Proteoglicanas de Sulfatos de Condroitina , Dendritos , Neurogênese , Neurônios , Transdução de Sinais
6.
Development ; 146(9)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30967426

RESUMO

Establishing apical-basal epithelial cell polarity is fundamental for mammary gland duct morphogenesis during mammalian development. While the focal adhesion adapter protein paxillin is a well-characterized regulator of mesenchymal cell adhesion signaling, F-actin cytoskeleton remodeling and single cell migration, its role in epithelial tissue organization and mammary gland morphogenesis in vivo has not been investigated. Here, using a newly developed paxillin conditional knockout mouse model with targeted ablation in the mammary epithelium, in combination with ex vivo three-dimensional organoid and acini cultures, we identify new roles for paxillin in the establishment of apical-basal epithelial cell polarity and lumen formation, as well as mammary gland duct diameter and branching. Paxillin is shown to be required for the integrity and apical positioning of the Golgi network, Par complex and the Rab11/MyoVb trafficking machinery. Paxillin depletion also resulted in reduced levels of apical acetylated microtubules, and rescue experiments with the HDAC6 inhibitor tubacin highlight the central role for paxillin-dependent regulation of HDAC6 activity and associated microtubule acetylation in controlling epithelial cell apical-basal polarity and tissue branching morphogenesis.


Assuntos
Polaridade Celular/fisiologia , Células Epiteliais/citologia , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Paxilina/metabolismo , Animais , Movimento Celular/genética , Movimento Celular/fisiologia , Polaridade Celular/genética , Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Camundongos , Microtúbulos/metabolismo , Morfogênese/genética , Morfogênese/fisiologia , Paxilina/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
7.
Mol Neurobiol ; 56(8): 5749-5762, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30674037

RESUMO

Dendritogenesis can be impaired by exposure to alcohol, and aspects of this impairment share phenotypic similarities to dendritic defects observed after blockade of the Reelin-Dab1 tyrosine kinase signaling pathway. In this study, we find that 10 min of alcohol exposure (400 mg/dL ethanol) by itself causes an unexpected increase in tyrosine phosphorylation of many proteins including Src and Dab1 that are essential downstream effectors of Reelin signaling. This increase in phosphotyrosine is dose-dependent and blockable by selective inhibitors of Src Family Kinases (SFKs). However, the response is transient, and phosphotyrosine levels return to baseline after 30 min of continuous ethanol exposure, both in vitro and in vivo. During this latter period, Src is inactivated and Reelin application cannot stimulate Dab1 phosphorylation. This suggests that ethanol initially activates but then silences the Reelin-Dab1 signaling pathway by brief activation and then sustained inactivation of SFKs. Time-lapse analyses of dendritic growth dynamics show an overall decrease in growth and branching compared to controls after ethanol-exposure that is similar to that observed with Reelin-deficiency. However, unlike Reelin-signaling disruptions, the dendritic filopodial speeds are decreased after ethanol exposure, and this decrease is associated with sustained dephosphorylation and activation of cofilin, an F-actin severing protein. These findings suggest that persistent Src inactivation coupled to cofilin activation may contribute to the dendritic disruptions observed with fetal alcohol exposure.


Assuntos
Dendritos/metabolismo , Etanol/toxicidade , Quinases da Família src/antagonistas & inibidores , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Ativação Enzimática/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Células HEK293 , Humanos , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteína Reelina , Serina Endopeptidases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Quinases da Família src/metabolismo
8.
Development ; 144(21): 4002-4014, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28935710

RESUMO

Paxillin and Hic-5 are homologous focal adhesion adaptor proteins that coordinate cytoskeletal rearrangements in response to integrin signaling, but their role(s) in cortical development are unknown. Here, we find that Hic-5-deficient mice are postnatal viable with normal cortical layering. Mice with a neural-specific deletion of paxillin are also postnatal viable, but show evidence of a cortical neuron migration delay that is evident pre- and perinatally, but is not detected at postnatal day 35 (P35). This phenotype is not modified by Hic-5 deficiency (double knockout). Specific deletion of paxillin in postmitotic neurons using Nex-Cre-mediated recombination as well as in utero electroporation of a Cre-expression construct identified a cell-autonomous requirement for paxillin in migrating neurons. Paxillin-deficient neurons have shorter leading processes that exhibited multiple swellings in comparison with control. Multiphoton imaging revealed that paxillin-deficient neurons migrate ∼30% slower than control neurons. This phenotype is similar to that produced by deletion of focal adhesion kinase (FAK), a signaling partner of paxillin, and suggests that paxillin and FAK function cell-autonomously to control migrating neuron morphology and speed during cortical development.


Assuntos
Movimento Celular , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Adesões Focais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Paxilina/metabolismo , Alelos , Animais , Movimento Celular/genética , Forma Celular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Integrases/metabolismo , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Camundongos Knockout , Células-Tronco Neurais/metabolismo , Especificidade de Órgãos , Paxilina/deficiência , Paxilina/genética
9.
J Neurochem ; 142(1): 89-102, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28419454

RESUMO

Despite the recent identification of over 40 missense heterozygous Reelin gene (RELN) mutations in autism spectrum disorder (ASD), none of these has been functionally characterized. Reelin is an integral signaling ligand for proper brain development and post-natal synapse function - properties likely disrupted in ASD patients. We find that the R2290C mutation, which arose de novo in an affected ASD proband, and other analogous mutations in arginine-amino acid-arginine domains reduce protein secretion. Closer analysis of RELN R2290C heterozygous neurospheres reveals up-regulation of Protein Disulfide Isomerase A1, best known as an endoplasmic reticulum-chaperone protein, which has been linked to neuronal pathology. This effect is recapitulated in a heterozygous RELN mouse mutant that is characterized by defective Reelin secretion. These findings suggest that both a deficiency in Reelin signaling and pathologic impairment of Reelin secretion may contribute to ASD risk.


Assuntos
Transtorno do Espectro Autista/genética , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Animais , Transtorno do Espectro Autista/metabolismo , Diferenciação Celular/genética , Cerebelo/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Isomerases de Dissulfetos de Proteínas/biossíntese , Edição de RNA , Proteína Reelina , Receptores X de Retinoides/biossíntese , Receptores X de Retinoides/genética
10.
J Neurosci ; 35(30): 10659-74, 2015 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-26224852

RESUMO

The mechanisms controlling cortical dendrite initiation and targeting are poorly understood. Multiphoton imaging of developing mouse cortex reveals that apical dendrites emerge by direct transformation of the neuron's leading process during the terminal phase of neuronal migration. During this ∼110 min period, the dendritic arbor increases ∼2.5-fold in size and migration arrest occurs below the first stable branch point in the developing arbor. This dendritic outgrowth is triggered at the time of leading process contact with the marginal zone (MZ) and occurs primarily by neurite extension into the extracellular matrix of the MZ. In reeler cortices that lack the secreted glycoprotein Reelin, a subset of neurons completed migration but then retracted and reorganized their arbor in a tangential direction away from the MZ soon after migration arrest. For these reeler neurons, the tangential oriented primary neurites were longer lived than the radially oriented primary neurites, whereas the opposite was true of wild-type (WT) neurons. Application of Reelin protein to reeler cortices destabilized tangential neurites while stabilizing radial neurites and stimulating dendritic growth in the MZ. Therefore, Reelin functions as part of a polarity signaling system that links dendritogenesis in the MZ with cellular positioning and cortical lamination. SIGNIFICANCE STATEMENT: Whether the apical dendrite emerges by transformation of the leading process of the migrating neuron or emerges de novo after migration is completed is unclear. Similarly, it is not clear whether the secreted glycoprotein Reelin controls migration and dendritic growth as related or separate processes. Here, multiphoton microscopy reveals the direct transformation of the leading process into the apical dendrite. This transformation is coupled to the successful completion of migration and neuronal soma arrest occurs below the first stable branch point of the nascent dendrite. Deficiency in Reelin causes the forming dendrite to avoid its normal target area and branch aberrantly, leading to improper cellular positioning. Therefore, this study links Reelin-dependent dendritogenesis with migration arrest and cortical lamination.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Dendritos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Neurogênese/fisiologia , Serina Endopeptidases/metabolismo , Animais , Western Blotting , Encéfalo/citologia , Encéfalo/metabolismo , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Células Cultivadas , Imuno-Histoquímica , Camundongos , Camundongos Mutantes Neurológicos , Microscopia Confocal , Proteína Reelina
11.
Mol Pharmacol ; 87(5): 825-31, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25710967

RESUMO

p53 is a Zn(2+)-dependent tumor suppressor inactivated in >50% of human cancers. The most common mutation, R175H, inactivates p53 by reducing its affinity for the essential zinc ion, leaving the mutant protein unable to bind the metal in the low [Zn(2+)]free environment of the cell. The exploratory cancer drug zinc metallochaperone-1 (ZMC1) was previously demonstrated to reactivate this and other Zn(2+)-binding mutants by binding Zn(2+) and buffering it to a level such that Zn(2+) can repopulate the defective binding site, but how it accomplishes this in the context of living cells and organisms is unclear. In this study, we demonstrated that ZMC1 increases intracellular [Zn(2+)]free by functioning as a Zn(2+) ionophore, binding Zn(2+) in the extracellular environment, diffusing across the plasma membrane, and releasing it intracellularly. It raises intracellular [Zn(2+)]free in cancer (TOV112D) and noncancer human embryonic kidney cell line 293 to 15.8 and 18.1 nM, respectively, with half-times of 2-3 minutes. These [Zn(2+)]free levels are predicted to result in ∼90% saturation of p53-R175H, thus accounting for its observed reactivation. This mechanism is supported by the X-ray crystal structure of the [Zn(ZMC1)2] complex, which demonstrates structural and chemical features consistent with those of known metal ionophores. These findings provide a physical mechanism linking zinc metallochaperone-1 in both in vitro and in vivo activities and define the remaining critical parameter necessary for developing synthetic metallochaperones for clinical use.


Assuntos
Transporte Biológico/fisiologia , Proteínas de Transporte/metabolismo , Ionóforos/metabolismo , Metalochaperonas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Zinco/metabolismo , Sítios de Ligação , Linhagem Celular , Membrana Celular/metabolismo , Células HEK293 , Humanos , Mutação/genética , Conformação Proteica , Proteína Supressora de Tumor p53/genética
12.
Front Pediatr ; 2: 121, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25426475

RESUMO

The development of the layered cerebral cortex starts with a process called preplate splitting. Preplate splitting involves the establishment of prospective cortical layer 6 (L6) neurons within a plexus of pioneer neurons called the preplate. The forming layer 6 splits the preplate into a superficial layer of pioneer neurons called the marginal zone and a deeper layer of pioneer neurons called the subplate. Disruptions of this early developmental event by toxin exposure or mutation are associated with neurological disease including severe intellectual disability. This review explores recent findings that reveal the dynamism of gene expression and morphological differentiation during this early developmental period. Over 1000 genes show expression increases of ≥2-fold during this period in differentiating mouse L6 neurons. Surprisingly, 88% of previously identified non-syndromic intellectual-disability (NS-ID) genes are expressed at this time and show an average expression increase of 1.6-fold in these differentiating L6 neurons. This changing genetic program must, in part, support the dramatic cellular reorganizations that occur during preplate splitting. While different models have been proposed for the formation of a layer of L6 cortical neurons within the preplate, original histological studies and more recent work exploiting transgenic mice suggest that the process is largely driven by the coordinated polarization and coalescence of L6 neurons rather than by cellular translocation or migration. The observation that genes associated with forms of NS-ID are expressed during very early cortical development raises the possibility of studying the relevant biological events at a time point when the cortex is small, contains relatively few cell types, and few functional circuits. This review then outlines how explant models may prove particularly useful in studying the consequence of toxin and mutation on the etiology of some forms of NS-ID.

13.
J Neurosci ; 34(2): 539-53, 2014 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-24403153

RESUMO

The three-layered piriform cortex, an integral part of the olfactory system, processes odor information relayed by olfactory bulb mitral cells. Specifically, mitral cell axons form the lateral olfactory tract (LOT) by targeting lateral olfactory tract (lot) guidepost cells in the piriform cortex. While lot cells and other piriform cortical neurons share a pallial origin, the factors that specify their precise phenotypes are poorly understood. Here we show that in mouse, the proneural genes Neurog1 and Neurog2 are coexpressed in the ventral pallium, a progenitor pool that first gives rise to Cajal-Retzius (CR) cells, which populate layer I of all cortical domains, and later to layer II/III neurons of the piriform cortex. Using loss-of-function and gain-of-function approaches, we find that Neurog1 has a unique early role in reducing CR cell neurogenesis by tempering Neurog2's proneural activity. In addition, Neurog1 and Neurog2 have redundant functions in the ventral pallium, acting in two phases to first specify a CR cell fate and later to specify layer II/III piriform cortex neuronal identities. In the early phase, Neurog1 and Neurog2 are also required for lot cell differentiation, which we reveal are a subset of CR neurons, the loss of which prevents mitral cell axon innervation and LOT formation. Consequently, mutation of Trp73, a CR-specific cortical gene, results in lot cell and LOT axon displacement. Neurog1 and Neurog2 thus have unique and redundant functions in the piriform cortex, controlling the timing of differentiation of early-born CR/lot cells and specifying the identities of later-born layer II/III neurons.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Córtex Cerebral/embriologia , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Neurônios/citologia , Animais , Diferenciação Celular/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Eletroporação , Embrião de Mamíferos , Feminino , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Mutantes , Células-Tronco Neurais/metabolismo
14.
PLoS One ; 8(8): e70962, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23951053

RESUMO

Metastasizing tumor cells undergo a transformation that resembles a process in normal development when non-migratory epithelial cells modulate the expression of cytoskeletal and adhesion proteins to promote cell motility. Here we find a mesenchymal cadherin, Cadherin-11 (CDH11), is increased in cells exiting the ventricular zone (VZ) neuroepithelium during normal cerebral cortical development. When overexpressed in cortical progenitors in vivo, CDH11 causes premature exit from the neuroepithelium and increased cell migration. CDH11 expression is elevated in human brain tumors, correlating with higher tumor grade and decreased patient survival. In glioblastoma, CDH11-expressing tumor cells can be found localized near tumor vasculature. Endothelial cells stimulate TGFß signaling and CDH11 expression in glioblastoma cells. TGFß promotes glioblastoma cell motility, and knockdown of CDH11 expression in primary human glioblastoma cells inhibits TGFß-stimulated migration. Together, these findings show that Cadherin-11 can promote cell migration in neural precursors and glioblastoma cells and suggest that endothelial cells increase tumor aggressiveness by co-opting mechanisms that regulate normal neural development.


Assuntos
Caderinas/genética , Movimento Celular/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neurais/metabolismo , Animais , Western Blotting , Caderinas/metabolismo , Movimento Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Feminino , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Neoplásicas/patologia , Células-Tronco Neurais/citologia , Gravidez , Interferência de RNA , Fator de Crescimento Transformador beta/farmacologia , Células Tumorais Cultivadas
15.
J Vis Exp ; (74)2013 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-23609059

RESUMO

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves -80% success in targeting GFP expression to neurons developing in the dorsal medial cortex. The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.


Assuntos
Encéfalo/embriologia , Eletroporação/métodos , Animais , DNA/administração & dosagem , DNA/genética , Feminino , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos , Plasmídeos/administração & dosagem , Plasmídeos/genética , Gravidez
16.
BMC Neurosci ; 13: 90, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-22852769

RESUMO

BACKGROUND: Cortical neurons display dynamic patterns of gene expression during the coincident processes of differentiation and migration through the developing cerebrum. To identify genes selectively expressed by the Eomes + (Tbr2) lineage of excitatory cortical neurons, GFP-expressing cells from Tg(Eomes::eGFP) Gsat embryos were isolated to > 99% purity and profiled. RESULTS: We report the identification, validation and spatial grouping of genes selectively expressed within the Eomes + cortical excitatory neuron lineage during early cortical development. In these neurons 475 genes were expressed ≥ 3-fold, and 534 genes ≤ 3-fold, compared to the reference population of neuronal precursors. Of the up-regulated genes, 328 were represented at the Genepaint in situ hybridization database and 317 (97%) were validated as having spatial expression patterns consistent with the lineage of differentiating excitatory neurons. A novel approach for quantifying in situ hybridization patterns (QISP) across the cerebral wall was developed that allowed the hierarchical clustering of genes into putative co-regulated groups. Forty four candidate genes were identified that show spatial expression with Intermediate Precursor Cells, 49 candidate genes show spatial expression with Multipolar Neurons, while the remaining 224 genes achieved peak expression in the developing cortical plate. CONCLUSIONS: This analysis of differentiating excitatory neurons revealed the expression patterns of 37 transcription factors, many chemotropic signaling molecules (including the Semaphorin, Netrin and Slit signaling pathways), and unexpected evidence for non-canonical neurotransmitter signaling and changes in mechanisms of glucose metabolism. Over half of the 317 identified genes are associated with neuronal disease making these findings a valuable resource for studies of neurological development and disease.


Assuntos
Linhagem da Célula/genética , Córtex Cerebral/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurogênese/genética , Animais , Diferenciação Celular/genética , Análise por Conglomerados , Bases de Dados Genéticas/estatística & dados numéricos , Citometria de Fluxo/métodos , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/estatística & dados numéricos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Neurônios/fisiologia , Proteínas com Domínio T/genética
17.
Alcohol ; 46(7): 619-27, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22840816

RESUMO

Prenatal ethanol exposure disrupts cortical neurite initiation and outgrowth, but prior studies have reported both ethanol-dependent growth promotion and inhibition. To resolve this ambiguity and better approximate in vivo conditions, we quantitatively analyzed neuronal morphology using a new, whole hemisphere explant model. In this model, Layer 6 (L6) cortical neurons migrate, laminate and extend neurites in an organotypic fashion. To selectively label L6 neurons, we performed ex utero electroporation of a GFP expression construct at embryonic day 13 and allowed the explants to develop for 2 days in vitro. Explants were exposed to (400 mg/dL) ethanol for either 4 or 24 h prior to fixation. Complete 3-D reconstructions were made of >80 GFP-positive neurons in each experimental condition. Acute responses to ethanol exposure included compaction of the Golgi apparatus accompanied by elaboration of supernumerary primary apical neurites, as well as a modest (∼15%) increase in higher order apical neurite length. With longer exposure time, ethanol exposure leads to a consistent, significant disorientation of the cell (cell body, primary apical neurite, and Golgi) with respect to the pial surface. The effects on cellular orientation were accompanied by decreased expression of cytoskeletal elements, microtubule-associated protein 2 and F-actin. These findings indicate that upon exposure to ethanol, developing L6 neurons manifest disruptions in Golgi apparatus and cytoskeletal elements which may in turn trigger selective and significant perturbations to primary neurite formation and neuronal polarity.


Assuntos
Polaridade Celular/efeitos dos fármacos , Cérebro/efeitos dos fármacos , Etanol/toxicidade , Complexo de Golgi/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Actinas/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Cérebro/embriologia , Cérebro/metabolismo , Cérebro/patologia , Dendritos/efeitos dos fármacos , Dendritos/patologia , Eletroporação , Feminino , Técnicas de Transferência de Genes , Idade Gestacional , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Imageamento Tridimensional , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/patologia , Neurônios/metabolismo , Neurônios/patologia , Técnicas de Cultura de Órgãos , Gravidez , Fatores de Tempo
18.
Neural Dev ; 7: 25, 2012 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-22770513

RESUMO

BACKGROUND: The secreted ligand Reelin is believed to regulate the translocation of prospective layer 6 (L6) neocortical neurons into the preplate, a loose layer of pioneer neurons that overlies the ventricular zone. Recent studies have also suggested that Reelin controls neuronal orientation and polarized dendritic growth during this period of early cortical development. To explicitly characterize and quantify how Reelin controls this critical aspect of neurite initiation and growth we used a new ex utero explant model of early cortical development to selectively label a subset of L6 cortical neurons for complete 3-D reconstruction. RESULTS: The total neurite arbor sizes of neurons in Reelin-deficient (reeler mutant) and Dab1-deficient (Reelin-non-responsive scrambler mutant) cortices were quantified and unexpectedly were not different than control arbor lengths (p = 0.51). For each mutant, however, arbor organization was markedly different: mutant neurons manifested more primary processes (neurites emitted directly from the soma) than wild type, and these neurites were longer and displayed less branching. Reeler and scrambler mutant neurites extended tangentially rather than radially, and the Golgi apparatus that normally invests the apical neurite was compact in both reeler and scrambler mutants. Mutant cortices also exhibited a neurite "exclusion zone" which was relatively devoid of L6 neuron neurites and extended at least 15 µm beneath the pial surface, an area corresponding to the marginal zone (MZ) in the wild type explants. The presence of an exclusion zone was also indicated in the orientation of mutant primary neurite and neuronal somata, which failed to adopt angles within ~20˚ of the radial line to the pial surface. Injection of recombinant Reelin to reeler, but not scrambler, mutant cortices fully rescued soma orientation, Golgi organization, and dendritic projection defects within four hrs. CONCLUSIONS: These findings indicate Reelin promotes directional dendritic growth into the MZ, an otherwise exclusionary zone for L6 neurites.


Assuntos
Moléculas de Adesão Celular Neuronais/genética , Proteínas da Matriz Extracelular/genética , Neocórtex/anormalidades , Neocórtex/citologia , Proteínas do Tecido Nervoso/genética , Neuritos/metabolismo , Neurônios/metabolismo , Serina Endopeptidases/genética , Animais , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/farmacologia , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/farmacologia , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Neocórtex/metabolismo , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/ultraestrutura , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Técnicas de Cultura de Órgãos , Gravidez , Proteína Reelina , Serina Endopeptidases/deficiência , Serina Endopeptidases/farmacologia
19.
Cell ; 143(5): 826-36, 2010 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-21111240

RESUMO

The Reelin ligand regulates a Dab1-dependent signaling pathway required for brain lamination and normal dendritogenesis, but the specific mechanisms underlying these actions remain unclear. We find that Stk25, a modifier of Reelin-Dab1 signaling, regulates Golgi morphology and neuronal polarization as part of an LKB1-Stk25-Golgi matrix protein 130 (GM130) signaling pathway. Overexpression of Stk25 induces Golgi condensation and multiple axons, both of which are rescued by Reelin treatment. Reelin stimulation of cultured neurons induces the extension of the Golgi into dendrites, which is suppressed by Stk25 overexpression. In vivo, Reelin and Dab1 are required for the normal extension of the Golgi apparatus into the apical dendrites of hippocampal and neocortical pyramidal neurons. This demonstrates that the balance between Reelin-Dab1 signaling and LKB1-Stk25-GM130 regulates Golgi dispersion, axon specification, and dendrite growth and provides insights into the importance of the Golgi apparatus for cell polarization.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Complexo de Golgi/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Serina Endopeptidases/metabolismo , Animais , Linhagem Celular , Separação Celular , Células Cultivadas , Hipocampo/metabolismo , Humanos , Camundongos , Ratos , Proteína Reelina
20.
Cereb Cortex ; 20(9): 2213-23, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20064940

RESUMO

The secreted ligand Reelin is thought to regulate the translocation and positioning of prospective layer 6 (L6) neurons into the preplate, a plexus of neurons overlying the ventricular zone. We examined wild type and Reelin-deficient cortices and found that L6 neurons were equivalently positioned beneath the pia during the period of preplate splitting and initial cortical plate (CP) formation. The absence of detectable L6 ectopia in "reeler" cortices at this developmental time point indicates that Reelin-signaling might not regulate L6 neuron migration or gross positioning during preplate splitting. To explore the acute response of L6 neurons to Reelin, subpial injections of Reelin were made into Reelin-deficient explants. Reelin injection caused L6 neurons to orient their nuclei and polarize their Golgi toward the pia while initiating exuberant dendritic (MAP2+) outgrowth within 4 h. This rapid Reelin-dependent neuronal orientation and alignment created CP-like histology without any significant change in the mean position of the population of L6 neurons. Conversely, subplate cells and chondroitin sulfate proteoglycan immunoreactivity were found at significantly deeper positions from the pial surface after injection, suggesting that Reelin partially rescues preplate splitting within 4 h. Thus, Reelin has a direct role in promoting rapid morphological differentation and orientation of L6 neurons during preplate splitting.


Assuntos
Moléculas de Adesão Celular Neuronais/fisiologia , Polaridade Celular/genética , Córtex Cerebral/embriologia , Dendritos/metabolismo , Proteínas da Matriz Extracelular/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurogênese/genética , Neurônios/metabolismo , Serina Endopeptidases/fisiologia , Animais , Moléculas de Adesão Celular Neuronais/genética , Diferenciação Celular/genética , Córtex Cerebral/citologia , Proteínas da Matriz Extracelular/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Técnicas de Cultura de Órgãos , Proteína Reelina , Serina Endopeptidases/genética
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