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1.
Cell Rep ; 24(3): 577-584, 2018 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-30021156

RESUMO

Escape from peripheral tolerance checkpoints that control cytotoxic CD8+ T cells is important for cancer immunotherapy and autoimmunity, but pathways enforcing these checkpoints are mostly uncharted. We reveal that the HECT-type ubiquitin ligase activator, NDFIP1, enforces a cell-intrinsic CD8+ T cell checkpoint that desensitizes TCR signaling during in vivo exposure to high antigen levels. Ndfip1-deficient OT-I CD8+ T cells responding to high exogenous tolerogenic antigen doses that normally induce anergy aberrantly expanded and differentiated into effector cells that could precipitate autoimmune diabetes in RIP-OVAhi mice. In contrast, NDFIP1 was dispensable for peripheral deletion to low-dose exogenous or pancreatic islet-derived antigen and had little impact upon effector responses to Listeria or acute LCMV infection. These data provide evidence that NDFIP1 mediates a CD8+ T cell tolerance checkpoint, with a different mechanism to CD4+ T cells, and indicates that CD8+ T cell deletion and anergy are molecularly separable checkpoints.

2.
Exp Neurol ; 295: 36-45, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28511841

RESUMO

The two neuronal populations in the cortex, pyramidal neurons and interneurons, can be separated based on neurotransmitter identity, however, within this segregation a large degree of diversity exists. Investigations into the molecular diversity of neurons are impeded by the inability to isolate cell populations born at different times for gene expression analysis. Developing interneurons may be distinguished by the expression of Glutamic Acid Decarboxylase-67 (GAD67). Neuronal birthdating using nucleoside analogs is an effective means of identifying coetaneous interneurons. Using these two features, neurotransmitter identity and birthdating, we have developed a method to isolate migrating interneurons using fluorescent-activated cell sorting (FACS) for RNA extraction and gene expression analysis. We utilized 5-ethynyl-2'-deoxyuridine (EdU) to birthdate interneuron cohorts and the GAD67 knock-in GFP transgenic mice to identify interneurons. In combination, we achieved simultaneous detection of GFP and EdU signals during FACS sorting of coetaneous interneurons with minimum loss of RNA integrity. RNA quality was deemed to be satisfactory by quantitative polymerase chain reaction (qPCR) for the interneuron-specific transcript Gad67.


Assuntos
Separação Celular/métodos , Córtex Cerebral/citologia , Expressão Gênica , Técnicas Genéticas , Interneurônios , Animais , Permeabilidade da Membrana Celular , Citometria de Fluxo/métodos , Regulação da Expressão Gênica no Desenvolvimento , Glutamato Descarboxilase/genética , Proteínas de Fluorescência Verde , Humanos , Camundongos , Camundongos Transgênicos , Células Piramidais , RNA/biossíntese , RNA/genética
3.
Mol Ther ; 25(6): 1269-1278, 2017 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-28412169

RESUMO

Exosomes represent an attractive vehicle for the delivery of biomolecules. However, mechanisms for loading functional molecules into exosomes are relatively unexplored. Here we report the use of the evolutionarily conserved late-domain (L-domain) pathway as a mechanism for loading exogenous proteins into exosomes. We demonstrate that labeling of a target protein, Cre recombinase, with a WW tag leads to recognition by the L-domain-containing protein Ndfip1, resulting in ubiquitination and loading into exosomes. Our results show that Ndfip1 expression acts as a molecular switch for exosomal packaging of WW-Cre that can be suppressed using the exosome inhibitor GW4869. When taken up by floxed reporter cells, exosomes containing WW-Cre were capable of inducing DNA recombination, indicating functional delivery of the protein to recipient cells. Engineered exosomes were administered to the brain of transgenic reporter mice using the nasal route to test for intracellular protein delivery in vivo. This resulted in the transport of engineered exosomes predominantly to recipient neurons in a number of brain regions, including the olfactory bulb, cortex, striatum, hippocampus, and cerebellum. The ability to engineer exosomes to deliver biologically active proteins across the blood-brain barrier represents an important step for the development of therapeutics to treat brain diseases.


Assuntos
Sistemas de Liberação de Medicamentos , Exossomos/metabolismo , Engenharia Genética , Transporte Proteico , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Vesículas Extracelulares/metabolismo , Expressão Gênica , Engenharia Genética/métodos , Integrases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Absorção Nasal , Permeabilidade , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
4.
Cereb Cortex ; 27(1): 576-588, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-26503265

RESUMO

Mutations of the reelin gene cause severe defects in cerebral cortex development and profound intellectual impairment. While many aspects of the reelin signaling pathway have been identified, the molecular and ultimate cellular consequences of reelin signaling remain unknown. Specifically, it is unclear if termination of reelin signaling is as important for normal cortical neuron migration as activation of reelin signaling. Using mice that are single or double deficient, we discovered that combined loss of the suppressors of cytokine signaling, SOCS6 and SOCS7, recapitulated the cortical layer inversion seen in mice lacking reelin and led to a dramatic increase in the reelin signaling molecule disabled (DAB1) in the cortex. The SRC homology domains of SOCS6 and SOCS7 bound DAB1 ex vivo. Mutation of DAB1 greatly diminished binding and protected from degradation by SOCS6. Phosphorylated DAB1 was elevated in cortical neurons in the absence of SOCS6 and SOCS7. Thus, constitutive activation of reelin signaling was observed to be equally detrimental as lack of activation. We hypothesize that, by terminating reelin signaling, SOCS6 and SOCS7 may allow new cycles of reelin signaling to occur and that these may be essential for cortical neuron migration.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/metabolismo , Proteínas Supressoras da Sinalização de Citocina/deficiência , Animais , Moléculas de Adesão Celular Neuronais/genética , Movimento Celular/fisiologia , Córtex Cerebral/patologia , Proteínas da Matriz Extracelular/genética , Células HEK293 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Fosforilação , Serina Endopeptidases/genética , Proteínas Supressoras da Sinalização de Citocina/genética
5.
Sci Rep ; 6: 29514, 2016 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-27404227

RESUMO

Copy number variations to chromosome 21 (HSA21) cause intellectual disability and Down Syndrome, but our understanding of the HSA21 genetic factors which contribute to fetal brain development remains incomplete. Here, we focussed on the neurodevelopmental functions for EURL (also known as C21ORF91, Refseq Gene ID:54149), a protein-coding gene at the centromeric boundary of the Down Syndrome Critical Region (DSCR) of HSA21. We report that EURL is expressed during human and mouse cerebral cortex development, and we report that alterations to EURL mRNA levels within the human brain underlie Down Syndrome. Our gene perturbation studies in mice demonstrate that disruptions to Eurl impair progenitor proliferation and neuronal differentiation. Also, we find that disruptions to Eurl impair the long-term positioning and dendritic spine densities of cortical projection neurons. We provide evidence that EURL interacts with the coiled-coil domain-containing protein CCDC85B so as to modulate ß-catenin levels in cells. Further, we utilised a fluorescent reporter (8xTOPFLASHd2EGFP) to demonstrate that disruptions to Eurl alter ß-catenin signalling in vitro as well as in vivo. Together, these studies highlight EURL as an important new player in neuronal development that is likely to impact on the neuropathogenesis of HSA21-related disorders including Down Syndrome.


Assuntos
Córtex Cerebral/embriologia , Cromossomos Humanos Par 21/genética , Síndrome de Down/genética , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Variações do Número de Cópias de DNA/genética , Espinhas Dendríticas/patologia , Modelos Animais de Doenças , Síndrome de Down/metabolismo , Síndrome de Down/patologia , Humanos , Deficiência Intelectual/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas Repressoras/metabolismo , beta Catenina/metabolismo
6.
J Mol Cell Biol ; 7(2): 119-31, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25801959

RESUMO

Pten controls a signaling axis that is implicated to regulate cell proliferation, growth, survival, migration, and metabolism. The molecular mechanisms underlying the specificity of Pten responses to such diverse cellular functions are currently poorly understood. Here we report the control of Pten activity and signaling specificity during the cell cycle by Ndfip1 regulation of Pten spatial distribution. Genetic deletion of Ndfip1 resulted in a loss of Pten nuclear compartmentalization and increased cell proliferation, despite cytoplasmic Pten remaining active in regulating PI3K/Akt signaling. Cells lacking nuclear Pten were found to have dysregulated levels of Plk1 and cyclin D1 that could drive cell proliferation. In vivo, transgene expression of Ndfip1 in the developing brain increased nuclear Pten and lengthened the cell cycle of neuronal progenitors, resulting in microencephaly. Our results show that local partitioning of Pten from the cytoplasm to the nucleus represents a key mechanism contributing to the specificity of Pten signaling during cell proliferation.


Assuntos
Proteínas de Transporte/fisiologia , Proliferação de Células , Proteínas de Membrana/fisiologia , PTEN Fosfo-Hidrolase/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Ciclina D1/metabolismo , Feminino , Indazóis/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Microcefalia/metabolismo , Células PC12 , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Transdução de Sinais , Sirolimo/farmacologia , Sulfonamidas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo
7.
J Biol Chem ; 290(11): 7141-50, 2015 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-25631046

RESUMO

During injury, cells are vulnerable to apoptosis from a variety of stress conditions including DNA damage causing double-stranded breaks. Without repair, these breaks lead to aberrations in DNA replication and transcription, leading to apoptosis. A major response to DNA damage is provided by the protein kinase ATM (ataxia telangiectasia mutated) that is capable of commanding a plethora of signaling networks for DNA repair, cell cycle arrest, and even apoptosis. A key element in the DNA damage response is the mobilization of activating proteins into the cell nucleus to repair damaged DNA. BRAT1 is one of these proteins, and it functions as an activator of ATM by maintaining its phosphorylated status while also keeping other phosphatases at bay. However, it is unknown how BRAT1 is trafficked into the cell nucleus to maintain ATM phosphorylation. Here we demonstrate that Ndfip1-mediated ubiquitination of BRAT1 leads to BRAT1 trafficking into the cell nucleus. Without Ndfip1, BRAT1 failed to translocate to the nucleus. Under genotoxic stress, cells showed increased expression of both Ndfip1 and phosphorylated ATM. Following brain injury, neurons show increased expression of Ndfip1 and nuclear translocation of BRAT1. These results point to Ndfip1 as a sensor protein during cell injury and Ndfip1 up-regulation as a cue for BRAT1 ubiquitination by Nedd4 E3 ligases, followed by nuclear translocation of BRAT1.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Lesões Encefálicas/metabolismo , Linhagem Celular , Dano ao DNA , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Ubiquitina-Proteína Ligases Nedd4 , Ligação Proteica , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteólise , Transdução de Sinais , Ubiquitinação
9.
Cereb Cortex ; 25(3): 806-16, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24084125

RESUMO

The zinc finger transcription factor RP58 (also known as ZNF238) regulates neurogenesis of the mouse neocortex and cerebellum (Okado et al. 2009; Xiang et al. 2011; Baubet et al. 2012; Ohtaka-Maruyama et al. 2013), but its mechanism of action remains unclear. In this study, we report a cell-autonomous function for RP58 during the differentiation of embryonic cortical projection neurons via its activities as a transcriptional repressor. Disruption of RP58 expression alters the differentiation of immature neurons and impairs their migration and positioning within the mouse cerebral cortex. Loss of RP58 within the embryonic cortex also leads to elevated mRNA for Rnd2, a member of the Rnd family of atypical RhoA-like GTPase proteins important for cortical neuron migration (Heng et al. 2008). Mechanistically, RP58 represses transcription of Rnd2 via binding to a 3'-regulatory enhancer in a sequence-specific fashion. Using reporter assays, we found that RP58 repression of Rnd2 is competed by proneural basic helix-loop-helix transcriptional activators. Finally, our rescue experiments revealed that negative regulation of Rnd2 by RP58 was important for cortical cell migration in vivo. Taken together, these studies demonstrate that RP58 is a key player in the transcriptional control of cell migration in the developing cerebral cortex.


Assuntos
Movimento Celular/genética , Córtex Cerebral/embriologia , Córtex Cerebral/fisiologia , Neurônios/metabolismo , Proteínas Repressoras/genética , Proteínas rho de Ligação ao GTP/genética , Animais , Proliferação de Células/genética , Córtex Cerebral/metabolismo , Feminino , Masculino , Camundongos , Camundongos Knockout
10.
Methods ; 77-78: 157-63, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25542098

RESUMO

PTEN was discovered as a membrane-associated tumor suppressor protein nearly two decades ago, but the concept that it can be secreted and taken up by recipient cells is revolutionary. Since then, various laboratories have reported that PTEN is indeed secreted and available for uptake by other cells in at least two different guises. First, PTEN may be packaged and exported within extracellular vesicles (EV) called exosomes. Second, PTEN may also be secreted as a naked protein in a longer isoform called PTEN-long. While the conditions favouring the secretion of PTEN-long remain unknown, PTEN secretion in exosomes is enhanced by the Ndfip1/Nedd4 ubiquitination system. In this report, we describe conditions for packaging PTEN in exosomes and their potential use for mediating non cell-autonomous functions in recipient cells. We suggest that this mode of PTEN transfer may potentially provide beneficial PTEN for tumor suppression, however it may also propagate deleterious versions of mutated PTEN causing tumorigenesis.


Assuntos
Exossomos/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos
11.
Sci Signal ; 7(332): pe15, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24985344

RESUMO

The tumor suppressor PTEN is a major brake for cell transformation, mainly due to its phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] phosphatase activity that directly counteracts the oncogenicity of phosphoinositide 3-kinase (PI3K). PTEN mutations are frequent in tumors and in the germ line of patients with tumor predisposition or with neurological or cognitive disorders, which makes the PTEN gene and protein a major focus of interest in current biomedical research. After almost two decades of intense investigation on the 403-residue-long PTEN protein, a previously uncharacterized form of PTEN has been discovered that contains 173 amino-terminal extra amino acids, as a result of an alternate translation initiation site. To facilitate research in the field and to avoid ambiguities in the naming and identification of PTEN amino acids from publications and databases, we propose here a unifying nomenclature and amino acid numbering for this longer form of PTEN.


Assuntos
Aminoácidos/química , Códon de Iniciação , Bases de Dados de Proteínas , PTEN Fosfo-Hidrolase/química , Sequência de Aminoácidos , Humanos , PTEN Fosfo-Hidrolase/genética , Terminologia como Assunto
12.
Traffic ; 15(7): 749-61, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24798731

RESUMO

The spatial regulation of Pten is critical for its role as a tumour suppressor with both nuclear and cytoplasmic locations being implicated with distinct functions. In the cytoplasm, Pten plays a central role in opposing PI3K/Akt cell signalling, whereas in the nucleus, Pten is important for maintaining genome stability and enhancing the tumour suppressor activity of APC-CDH1. Despite this diversity in protein function at different subcellular locations, there is limited knowledge on how Pten is able to find different cellular niches. Here, we report that Rab5 GTPase is required for efficient trafficking and ubiquitination of Pten on endosomes inside the cytosol. Using bimolecular fluorescence complementation (BiFC) for imaging protein interactions, we observed that ubiquitinated Pten is localized to peri-nuclear and nuclear regions of the cell. Nuclear trafficking of Pten required both Rab5 as well as the E3 ligase adaptor protein Ndfip1. Rab5 colocalization with Pten was observed on endosomes and expression of a dominant negative form of Rab5 significantly reduced Pten ubiquitination and nuclear trafficking. Genomic deletion of Ndfip1 abrogated nuclear trafficking of ubiquitinated Pten, even in the presence of Rab5. Our findings show that endosomal trafficking and ubiquitination are important mechanisms for the subcellular distribution of Pten.


Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Proteínas de Membrana/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Endossomos/metabolismo , Camundongos , Transporte Proteico , Ubiquitinação
13.
Proc Natl Acad Sci U S A ; 111(6): 2067-74, 2014 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-24520172

RESUMO

The NDFIP1 (neural precursor cell expressed, developmentally down-regulated protein 4 family-interacting protein 1) adapter for the ubiquitin ligase ITCH is genetically linked to human allergic and autoimmune disease, but the cellular mechanism by which these proteins enable foreign and self-antigens to be tolerated is unresolved. Here, we use two unique mouse strains--an Ndfip1-YFP reporter and an Ndfip1-deficient strain--to show that Ndfip1 is progressively induced during T-cell differentiation and activation in vivo and that its deficiency causes a cell-autonomous, Forkhead box P3-independent failure of peripheral CD4(+) T-cell tolerance to self and exogenous antigen. In small cohorts of antigen-specific CD4(+) cells responding in vivo, Ndfip1 was necessary for tolerogen-reactive T cells to exit cell cycle after one to five divisions and to abort Th2 effector differentiation, defining a step in peripheral tolerance that provides insights into the phenomenon of T-cell anergy in vivo and is distinct from the better understood process of Bcl2-interacting mediator of cell death-mediated apoptosis. Ndfip1 deficiency precipitated autoimmune pancreatic destruction and diabetes; however, this depended on a further accumulation of nontolerant anti-self T cells from strong stimulation by exogenous tolerogen. These findings illuminate a peripheral tolerance checkpoint that aborts T-cell clonal expansion against allergens and autoantigens and demonstrate how hypersensitive responses to environmental antigens may trigger autoimmunity.


Assuntos
Adaptação Fisiológica , Linfócitos T CD4-Positivos/imunologia , Proteínas de Transporte/fisiologia , Ciclo Celular , Proteínas de Membrana/fisiologia , Animais , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , Proliferação de Células , Fatores de Transcrição Forkhead/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL
14.
PLoS One ; 9(1): e87119, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24475238

RESUMO

Iron misregulation is a central component in the neuropathology of Parkinson's disease. The iron transport protein DMT1 is known to be increased in Parkinson's brains linking functional transport mechanisms with iron accumulation. The regulation of DMT1 is therefore critical to the management of iron uptake in the disease setting. We previously identified post-translational control of DMT1 levels through a ubiquitin-mediated pathway led by Ndfip1, an adaptor for Nedd4 family of E3 ligases. Here we show that loss of Ndfip1 from mouse dopaminergic neurons resulted in misregulation of DMT1 levels and increased susceptibility to iron induced death. We report that in human Parkinson's brains increased iron concentrations in the substantia nigra are associated with upregulated levels of Ndfip1 in dopaminergic neurons containing α-synuclein deposits. Additionally, Ndfip1 was also found to be misexpressed in astrocytes, a cell type normally devoid of this protein. We suggest that in Parkinson's disease, increased iron levels are associated with increased Ndfip1 expression for the regulation of DMT1, including abnormal Ndfip1 activation in non-neuronal cell types such as astrocytes.


Assuntos
Astrócitos/metabolismo , Proteínas de Transporte/metabolismo , Neurônios Dopaminérgicos/metabolismo , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Doença de Parkinson/genética , Substância Negra/metabolismo , Fatores de Transcrição/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Proteínas de Transporte/genética , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica , Humanos , Transporte de Íons , Ferro/farmacologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Cultura Primária de Células , Transdução de Sinais , Substância Negra/patologia , Fatores de Transcrição/genética , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
15.
Cereb Cortex ; 24(12): 3289-300, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23897647

RESUMO

Ubiquitin ligases of the Nedd4 family are important for axon and dendrite development, but little is known about their adaptor, Nedd4 family-interacting protein 1 (Ndfip1), that is responsible for their enzymatic activation. To study the function of Ndfip1 in cortical development, we generated a conditional knock-out (conditional KO) in neurons. The Ndfip1 conditional KO mice were viable; however, cortical neurons in the adult brain exhibited atrophic characteristics, including stunted dendritic arbors, blebbing of dendrites, and fewer dendritic spines. In electron micrographs, these neurons appeared shrunken with compacted somata and involutions of the nuclear membrane. In culture, Ndfip1 KO neurons exhibited exuberant sprouting suggesting loss of developmental control. Biochemical analysis of postsynaptic density (PSD) fractions from Ndfip1 KO cortical and hippocampal neurons showed that the postsynaptic proteins (Arc and PSD-95) were reduced compared with wild-type controls. In addition, the PI3 kinase/Akt signaling pathway was altered. These results indicate that Ndfip1, through its Nedd4 effectors, is important for the development of dendrites and dendritic spines in the cortex.


Assuntos
Proteínas de Transporte/genética , Espinhas Dendríticas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Membrana/genética , Neocórtex , Células Piramidais/diagnóstico por imagem , Animais , Animais Recém-Nascidos , Fracionamento Celular , Células Cultivadas , Proteína 4 Homóloga a Disks-Large , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Guanilato Quinases/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neocórtex/citologia , Neocórtex/embriologia , Neocórtex/crescimento & desenvolvimento , Nestina/genética , Nestina/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Ultrassonografia
16.
Cereb Cortex ; 24(11): 2835-47, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23749873

RESUMO

Malformations of cortical development can arise when projection neurons generated in the germinal zones fail to migrate properly into the cortical plate. This process is critically dependent on the Reelin glycoprotein, which when absent leads to an inversion of cortical layers and blurring of borders. Reelin has other functions including supporting neuron migration and maintaining their trajectories; however, the precise role on glial fiber-dependent or -independent migration of neurons remains controversial. In this study, we wish to test the hypothesis that migrating cortical neurons at different levels of the cortical wall have differential responses to Reelin. We exposed neurons migrating across the cortical wall to exogenous Reelin and monitored their migratory behavior using time-lapse imaging. Our results show that, in the germinal zones, exogenous Reelin retarded neuron migration and altered their trajectories. This behavior is in contrast to the response of neurons located in the intermediate zone (IZ), possibly because Reelin receptors are not expressed in this zone. In the reeler cortex, Reelin receptors are expressed in the IZ and exposure to exogenous Reelin was able to rescue the migratory defect. These studies demonstrate that migrating neurons have nonequivalent responses to Reelin depending on their location within the cortical wall.


Assuntos
Moléculas de Adesão Celular Neuronais/farmacologia , Movimento Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Proteínas da Matriz Extracelular/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Serina Endopeptidases/farmacologia , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Fatores Etários , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Análise de Variância , Animais , Linhagem Celular Transformada , Movimento Celular/genética , Eletroporação , Embrião de Mamíferos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Técnicas In Vitro , Camundongos , Camundongos Mutantes Neurológicos , Microscopia Confocal , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Técnicas de Cultura de Órgãos , Transfecção
17.
Exp Neurol ; 252: 37-46, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24275527

RESUMO

There is controversy whether accumulation of the tumor suppressor PTEN protein in the cell nucleus under stress conditions such as trauma and stroke causes cell death. A number of in vitro studies have reported enhanced apoptosis in neurons possessing nuclear PTEN, with the interpretation that its nuclear phosphatase activity leads to reduction of the survival protein phospho-Akt. However, there have been no in vivo studies to show that nuclear PTEN in neurons under stress is detrimental. Using a mouse model of injury, we demonstrate here that brain trauma altered the nucleo-cytoplasmic distribution of Pten, resulting in increased nuclear Pten but only in surviving neurons near the lesion. This event was driven by Ndfip1, an adaptor and activator of protein ubiquitination by Nedd4 E3 ligases. Neurons next to the lesion with nuclear PTEN were invariably negative for TUNEL, a marker for cell death. These neurons also showed increased Ndfip1 which we previously showed to be associated with neuron survival. Biochemical assays revealed that overall levels of Pten in the affected cortex were unchanged after trauma, suggesting that Pten abundance globally had not increased but rather Pten subcellular location in affected neurons had changed. Following experimental injury, the number of neurons with nuclear Pten was reduced in heterozygous mice (Ndfip1(+/-)) although lesion volumes were increased. We conclude that nuclear trafficking of Pten following injury leads to neuron survival not death.


Assuntos
Lesões Encefálicas/patologia , Núcleo Celular/metabolismo , Regulação da Expressão Gênica/fisiologia , Neurônios , PTEN Fosfo-Hidrolase/metabolismo , Análise de Variância , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/fisiologia , Proteínas de Transporte/genética , Sobrevivência Celular/genética , Citoplasma , Modelos Animais de Doenças , Lateralidade Funcional , Imunoprecipitação , Marcação In Situ das Extremidades Cortadas , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/metabolismo , Neurônios/patologia , Neurônios/ultraestrutura , Proteína Oncogênica v-akt , PTEN Fosfo-Hidrolase/genética , Transporte Proteico/genética
18.
Int J Dev Neurosci ; 33: 62-80, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24368173

RESUMO

This study characterizes the developmental patterns of seven key amino acids: glutamate, γ-amino-butyric acid (GABA), glycine, glutamine, aspartate, alanine and taurine in the mouse retina. We analyze amino acids in specific bipolar, amacrine and ganglion cell sub-populations (i.e. GABAergic vs. glycinergic amacrine cells) and anatomically distinct regions of photoreceptors and Müller cells (i.e. cell bodies vs. endfeet) by extracting data from previously described pattern recognition analysis. Pattern recognition statistically classifies all cells in the retina based on their neurochemical profile and surpasses the previous limitations of anatomical and morphological identification of cells in the immature retina. We found that the GABA and glycine cellular content reached adult-like levels in most neurons before glutamate. The metabolic amino acids glutamine, aspartate and alanine also reached maturity in most retinal cells before eye opening. When the overall amino acid profiles were considered for each cell group, ganglion cells and GABAergic amacrine cells matured first, followed by glycinergic amacrine cells and finally bipolar cells. Photoreceptor cell bodies reached adult-like amino acid profiles at P7 whilst Müller cells acquired typical amino acid profiles in their cell bodies at P7 and in their endfeet by P14. We further compared the amino acid profiles of the C57Bl/6J mouse with the transgenic X-inactivation mouse carrying the lacZ gene on the X chromosome and validated this animal model for the study of normal retinal development. This study provides valuable insight into normal retinal neurochemical maturation and metabolism and benchmark amino acid values for comparison with retinal disease, particularly those which occur during development.


Assuntos
Aminoácidos/metabolismo , Retina/crescimento & desenvolvimento , Retina/metabolismo , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Feminino , Óperon Lac/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/metabolismo , Retina/citologia , Vias Visuais/metabolismo
19.
Exp Neurol ; 250: 74-93, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24041986

RESUMO

Pattern recognition has been used for the complete and statistically rigid classification of retinal neurons in vertebrates such as the adult cat, primate, rat and goldfish. Here, we label the mouse retina with antibodies against seven amino acids and use pattern recognition to characterize distinct retinal neurochemical cell classes based on their unique amino acid signatures. We followed the development of the cell classes in the X-inactivation transgenic mouse expressing the lacZ reporter gene on one X-chromosome. This mouse allows clonally related cells to be identified through differential ß-galactosidase activity due to random X-chromosome inactivation. Pattern recognition analysis partitioned the retina into nine neuronal classes at birth, increasing to 19 classes at eye opening and 26 classes by adulthood. Emergence of new cell classes was partly attributed to new neuron types and partly to the splitting of classes from early ages from refinement of their amino acid profiles. All six GABAergic amacrine cell classes and most ganglion cell classes appeared by P7 whilst all the glycinergic amacrine cell classes did not appear till adulthood. Separable bipolar cell classes were not detected till eye opening. Photoreceptor cell classes were detected at P3 but inner and outer segments did not form separable classes until adulthood. More importantly, we show that cells which share common amino acid profiles also shared cell dispersion patterns. GABAergic amacrine cell classes with conventional and displaced counterparts transgressed clonal boundaries whereas GABAergic amacrine cell classes found exclusively in the inner nuclear layer and all glycinergic amacrine cell classes did not transgress. Ganglion cells displayed both dispersion patterns. This study provides a comprehensive neurochemical atlas of the developing mouse retina, tracking the amino acid levels within distinct neuronal populations and highlighting unique migratory patterns within subpopulations of inner retinal neurons.


Assuntos
Reconhecimento Automatizado de Padrão/métodos , Retina/citologia , Retina/metabolismo , Neurônios Retinianos/classificação , Neurônios Retinianos/metabolismo , Aminoácidos/análise , Animais , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Retina/crescimento & desenvolvimento , Neurônios Retinianos/citologia
20.
Neurosci Lett ; 555: 225-30, 2013 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-24036464

RESUMO

During development, protein substrates need to be removed and degraded when they are no longer required. The E3 ubiquitin ligases, including Nedd4 family proteins, are a major group of enzymes responsible for adding ubiquitin chains to protein substrates prior to their degradation. Ndfip1 (Nedd4 family-interacting protein 1) is an adaptor and activator for Nedd4-family ubiquitin ligases for increasing substrate specificity. To study Nedd4-mediated ubiquitination during cortical development, we have mapped the spatio-temporal dynamics of Ndfip1 protein expression by immunocytochemistry. Ndfip1 expression was observed from embryonic day 11 (E11.5) until adult stages. Its presence increased during the postnatal stages and peaked at postnatal day 7 (P7). Spatially, Ndfip1 was found in the ventricular and marginal zones during corticogenesis but also in the cortical plate and subplate during midstage cortical development (E15.5). Postnatally, Ndfip1 was expressed in all cortical neurons (but not in glial cells) and this expression was both ubiquitous and uniform across cortical layers involving both pyramidal and non-pyramidal neurons. This consistent but dynamic pattern of Ndfip1 expression in temporal and spatial domains of the cortical landscape is indicative of complex programs of protein ubiquitination during corticogenesis.


Assuntos
Proteínas de Transporte/metabolismo , Córtex Cerebral/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Interneurônios/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese , Ubiquitina-Proteína Ligases Nedd4 , Células Piramidais/metabolismo , Ubiquitinação
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