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1.
Cell ; 139(2): 299-311, 2009 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-19837033

RESUMO

Mutations in the genes encoding the Wnt receptor Frizzled-4 (FZD4), coreceptor LRP5, or the ligand Norrin disrupt retinal vascular development and cause ophthalmic diseases. Although Norrin is structurally unrelated to Wnts, it binds FZD4 and activates the canonical Wnt pathway. Here we show that the tetraspanin Tspan12 is expressed in the retinal vasculature, and loss of Tspan12 phenocopies defects seen in Fzd4, Lrp5, and Norrin mutant mice. In addition, Tspan12 genetically interacts with Norrin or Lrp5. Overexpressed TSPAN12 associates with the Norrin-receptor complex and significantly increases Norrin/beta-catenin but not Wnt/beta-catenin signaling, whereas Tspan12 siRNA abolishes transcriptional responses to Norrin but not Wnt3A in retinal endothelial cells. Signaling defects caused by Norrin or FZD4 mutations that are predicted to impair receptor multimerization are rescued by overexpression of TSPAN12. Our data indicate that Norrin multimers and TSPAN12 cooperatively promote multimerization of FZD4 and its associated proteins to elicit physiological levels of signaling.


Assuntos
Receptores Frizzled/metabolismo , Proteínas de Membrana/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Retina/embriologia , Transdução de Sinais , beta Catenina/metabolismo , Animais , Diterpenos , Células Endoteliais/metabolismo , Receptores Frizzled/genética , Humanos , Camundongos , Receptores Acoplados a Proteínas G/genética , Tetraspaninas , beta Catenina/genética
2.
Proc Natl Acad Sci U S A ; 115(50): E11827-E11836, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30478038

RESUMO

ß-Catenin signaling controls the development and maintenance of the blood-brain barrier (BBB) and the blood-retina barrier (BRB), but the division of labor and degree of redundancy between the two principal ligand-receptor systems-the Norrin and Wnt7a/Wnt7b systems-are incompletely defined. Here, we present a loss-of-function genetic analysis of postnatal BBB and BRB maintenance in mice that shows striking threshold and partial redundancy effects. In particular, the combined loss of Wnt7a and Norrin or Wnt7a and Frizzled4 (Fz4) leads to anatomically localized BBB defects that are far more severe than observed with loss of Wnt7a, Norrin, or Fz4 alone. In the cerebellum, selective loss of Wnt7a in glia combined with ubiquitous loss of Norrin recapitulates the phenotype observed with ubiquitous loss of both Wnt7a and Norrin, implying that glia are the source of Wnt7a in the cerebellum. Tspan12, a coactivator of Norrin signaling in the retina, is also active in BBB maintenance but is less potent than Norrin, consistent with a model in which Tspan12 enhances the amplitude of the Norrin signal in vascular endothelial cells. Finally, in the context of a partially impaired Norrin system, the retina reveals a small contribution to BRB development from the Wnt7a/Wnt7b system. Taken together, these experiments define the extent of CNS region-specific cooperation for several components of the Norrin and Wnt7a/Wnt7b systems, and they reveal substantial regional heterogeneity in the extent to which partially redundant ligands, receptors, and coactivators maintain the BBB and BRB.


Assuntos
Barreira Hematoencefálica/crescimento & desenvolvimento , Barreira Hematoencefálica/fisiologia , Barreira Hematorretiniana/crescimento & desenvolvimento , Barreira Hematorretiniana/fisiologia , Proteínas do Olho/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Wnt/fisiologia , Animais , Barreira Hematoencefálica/citologia , Barreira Hematorretiniana/citologia , Técnicas de Cultura de Células , Proteínas do Olho/genética , Receptores Frizzled/deficiência , Receptores Frizzled/genética , Receptores Frizzled/fisiologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Modelos Neurológicos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Tetraspaninas/deficiência , Tetraspaninas/genética , Tetraspaninas/fisiologia , Proteínas Wnt/deficiência , Proteínas Wnt/genética , beta Catenina/fisiologia
3.
Arterioscler Thromb Vasc Biol ; 38(11): 2691-2705, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30354230

RESUMO

Objective- Blood-CNS (central nervous system) barrier defects are implicated in retinopathies, neurodegenerative diseases, stroke, and epilepsy, yet, the pathological mechanisms downstream of barrier defects remain incompletely understood. Blood-retina barrier (BRB) formation and retinal angiogenesis require ß-catenin signaling induced by the ligand norrin (NDP [Norrie disease protein]), the receptor FZD4 (frizzled 4), coreceptor LRP5 (low-density lipoprotein receptor-like protein 5), and the tetraspanin TSPAN12 (tetraspanin 12). Impaired NDP/FZD4 signaling causes familial exudative vitreoretinopathy, which may lead to blindness. This study seeked to define cell type-specific functions of TSPAN12 in the retina. Approach and Results- A loxP-flanked Tspan12 allele was generated and recombined in endothelial cells using a tamoxifen-inducible Cdh5-CreERT2 driver. Resulting phenotypes were documented using confocal microscopy. RNA-Seq, histopathologic analysis, and electroretinogram were performed on retinas of aged mice. We show that TSPAN12 functions in endothelial cells to promote vascular morphogenesis and BRB formation in developing mice and BRB maintenance in adult mice. Early loss of TSPAN12 in endothelial cells causes lack of intraretinal capillaries and increased VE-cadherin (CDH5 [cadherin5 aka VE-cadherin]) expression, consistent with premature vascular quiescence. Late loss of TSPAN12 strongly impairs BRB maintenance without affecting vascular morphogenesis, pericyte coverage, or perfusion. Long-term BRB defects are associated with immunoglobulin extravasation, complement deposition, cystoid edema, and impaired b-wave in electroretinograms. RNA-sequencing reveals transcriptional responses to the perturbation of the BRB, including genes involved in vascular basement membrane alterations in diabetic retinopathy. Conclusions- This study establishes mice with late endothelial cell-specific loss of Tspan12 as a model to study pathological consequences of BRB impairment in an otherwise intact vasculature.


Assuntos
Barreira Hematorretiniana/metabolismo , Células Endoteliais/metabolismo , Neovascularização Retiniana , Vasos Retinianos/metabolismo , Tetraspaninas/deficiência , Fatores Etários , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Membrana Basal/metabolismo , Membrana Basal/patologia , Barreira Hematorretiniana/imunologia , Barreira Hematorretiniana/patologia , Caderinas/genética , Caderinas/metabolismo , Proliferação de Células , Senescência Celular , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Células Endoteliais/imunologia , Células Endoteliais/patologia , Oftalmopatias Hereditárias/genética , Oftalmopatias Hereditárias/metabolismo , Oftalmopatias Hereditárias/patologia , Vitreorretinopatias Exsudativas Familiares , Feminino , Genótipo , Imunoglobulinas/imunologia , Imunoglobulinas/metabolismo , Edema Macular/genética , Edema Macular/metabolismo , Edema Macular/patologia , Masculino , Camundongos Knockout , Fenótipo , Doenças Retinianas/genética , Doenças Retinianas/metabolismo , Doenças Retinianas/patologia , Vasos Retinianos/imunologia , Vasos Retinianos/patologia , Transdução de Sinais , Tetraspaninas/genética
4.
Cell Rep ; 42(5): 112455, 2023 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-37149867

RESUMO

Dynamic and coordinated axonal responses to changing environments are critical for establishing neural connections. As commissural axons migrate across the CNS midline, they are suggested to switch from being attracted to being repelled in order to approach and to subsequently leave the midline. A molecular mechanism that is hypothesized to underlie this switch in axonal responses is the silencing of Netrin1/Deleted in Colorectal Carcinoma (DCC)-mediated attraction by the repulsive SLIT/ROBO1 signaling. Using in vivo approaches including CRISPR-Cas9-engineered mouse models of distinct Dcc splice isoforms, we show here that commissural axons maintain responsiveness to both Netrin and SLIT during midline crossing, although likely at quantitatively different levels. In addition, full-length DCC in collaboration with ROBO3 can antagonize ROBO1 repulsion in vivo. We propose that commissural axons integrate and balance the opposing DCC and Roundabout (ROBO) signaling to ensure proper guidance decisions during midline entry and exit.


Assuntos
Proteínas do Tecido Nervoso , Receptores Imunológicos , Animais , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Orientação de Axônios , Axônios/metabolismo , Netrinas , Regulação da Expressão Gênica no Desenvolvimento , Receptor DCC
5.
iScience ; 26(8): 107415, 2023 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-37559903

RESUMO

Norrin (NDP) and WNT7A/B induce and maintain the blood-brain and blood-retina barrier (BBB, BRB) by stimulating the Frizzled4-LDL receptor related protein 5/6 (FZD4-LRP5/6) complex to induce beta-catenin-dependent signaling in endothelial cells (ECs). Recently developed agonists for the FZD4-LRP5 complex have therapeutic potential in retinal and neurological diseases. Here, we use the tetravalent antibody modality F4L5.13 to identify agonist activities in Tspan12-/- mice, which display a complex retinal pathology due to impaired NDP-signaling. F4L5.13 administration during development alleviates BRB defects, retinal hypovascularization, and restores neural function. In mature Tspan12-/- mice F4L5.13 partially induces a BRB de novo without inducing angiogenesis. In a genetic model of impaired BRB maintenance, administration of F4L5.13 rapidly and substantially restores the BRB. scRNA-seq reveals perturbations of key mediators of barrier functions in juvenile Tspan12-/- mice, which are in large parts restored after F4L5.13 administration. This study identifies transcriptional and functional activities of FZD4-LRP5 agonists.

6.
EMBO Mol Med ; 13(7): e13977, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34105895

RESUMO

The FZD4:LRP5:TSPAN12 receptor complex is activated by the secreted protein Norrin in retinal endothelial cells and leads to ßcatenin-dependent formation of the blood-retina-barrier during development and its homeostasis in adults. Mutations disrupting Norrin signaling have been identified in several congenital diseases leading to hypovascularization of the retina and blindness. Here, we developed F4L5.13, a tetravalent antibody designed to induce FZD4 and LRP5 proximity in such a way as to trigger ßcatenin signaling. Treatment of cultured endothelial cells with F4L5.13 rescued permeability induced by VEGF in part by promoting surface expression of junction proteins. Treatment of Tspan12-/- mice with F4L5.13 restored retinal angiogenesis and barrier function. F4L5.13 treatment also significantly normalized neovascularization in an oxygen-induced retinopathy model revealing a novel therapeutic strategy for diseases characterized by abnormal angiogenesis and/or barrier dysfunction.


Assuntos
Células Endoteliais , Doenças Retinianas , Animais , Barreira Hematorretiniana , Camundongos , Retina , Transdução de Sinais
7.
J Neurosci ; 28(8): 1949-60, 2008 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-18287511

RESUMO

Ca2+ regulates multiple processes in nerve terminals, including synaptic vesicle recruitment, priming, and fusion. Munc13s, the mammalian homologs of Caenorhabditis elegans Unc13, are essential vesicle-priming proteins and contain multiple regulatory domains that bind second messengers such as diacylglycerol and Ca2+/calmodulin (Ca2+/CaM). Binding of Ca2+/CaM is necessary for the regulatory effect that allows Munc13-1 and ubMunc13-2 to promote short-term synaptic plasticity. However, the relative contributions of Ca2+ and Ca2+/CaM to vesicle priming and recruitment by Munc13 are not known. Here, we investigated the effect of Ca2+/CaM binding on ubMunc13-2 activity in chromaffin cells via membrane-capacitance measurements and a detailed simulation of the exocytotic machinery. Stimulating secretion under various basal Ca2+ concentrations from cells overexpressing either ubMunc13-2 or a ubMunc13-2 mutant deficient in CaM binding enabled a distinction between the effects of Ca2+ and Ca2+/CaM. We show that vesicle priming by ubMunc13-2 is Ca2+ dependent but independent of CaM binding to ubMunc13-2. However, Ca2+/CaM binding to ubMunc13-2 specifically promotes vesicle recruitment during ongoing stimulation. Based on the experimental data and our simulation, we propose that ubMunc13-2 is activated by two Ca2+-dependent processes: a slow activation mode operating at low Ca2+ concentrations, in which ubMunc13-2 acts as a priming switch, and a fast mode at high Ca2+ concentrations, in which ubMunc13-2 is activated in a Ca2+/CaM-dependent manner and accelerates vesicle recruitment and maturation during stimulation. These different Ca2+ activation steps determine the kinetic properties of exocytosis and vesicle recruitment and can thus alter plasticity and efficacy of transmitter release.


Assuntos
Cálcio/fisiologia , Calmodulina/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Recrutamento Neurofisiológico/fisiologia , Vesículas Sinápticas/fisiologia , Animais , Bovinos , Células Cultivadas , Células Cromafins/citologia , Células Cromafins/metabolismo , Exocitose/fisiologia , Ligação Proteica/fisiologia , Vesículas Sinápticas/metabolismo
8.
Elife ; 82019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31392959

RESUMO

Proper connectivity of the nervous system requires temporal and spatial control of axon guidance signaling. As commissural axons navigate across the CNS midline, ROBO-mediated repulsion has traditionally been thought to be repressed before crossing, and then to become upregulated after crossing. The regulation of the ROBO receptors involves multiple mechanisms that control protein expression, trafficking, and activity. Here, we report that mammalian ROBO1 and ROBO2 are not uniformly inhibited precrossing and are instead subject to additional temporal control via alternative splicing at a conserved microexon. The NOVA splicing factors regulate the developmental expression of ROBO1 and ROBO2 variants with small sequence differences and distinct guidance activities. As a result, ROBO-mediated axonal repulsion is activated early in development to prevent premature crossing and becomes inhibited later to allow crossing. Postcrossing, the ROBO1 and ROBO2 isoforms are disinhibited to prevent midline reentry and to guide postcrossing commissural axons to distinct mediolateral positions.


Assuntos
Processamento Alternativo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/biossíntese , Crescimento Neuronal , Receptores Imunológicos/biossíntese , Animais , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Receptores Imunológicos/genética , Proteínas Roundabout
9.
Nat Commun ; 10(1): 5243, 2019 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-31748531

RESUMO

Familial exudative vitreoretinopathy (FEVR) is a human disease characterized by defective retinal angiogenesis and associated complications that can result in vision loss. Defective Wnt/ß-catenin signaling is an established cause of FEVR, whereas other molecular alterations contributing to the disease remain insufficiently understood. Here, we show that integrin-linked kinase (ILK), a mediator of cell-matrix interactions, is indispensable for retinal angiogenesis. Inactivation of the murine Ilk gene in postnatal endothelial cells results in sprouting defects, reduced endothelial proliferation and disruption of the blood-retina barrier, resembling phenotypes seen in established mouse models of FEVR. Retinal vascularization defects are phenocopied by inducible inactivation of the gene for α-parvin (Parva), an interactor of ILK. Screening genomic DNA samples from exudative vitreoretinopathy patients identifies three distinct mutations in human ILK, which compromise the function of the gene product in vitro. Together, our data suggest that defective cell-matrix interactions are linked to Wnt signaling and FEVR.


Assuntos
Barreira Hematorretiniana/metabolismo , Células Endoteliais/metabolismo , Vitreorretinopatias Exsudativas Familiares/genética , Neovascularização Fisiológica/genética , Proteínas Serina-Treonina Quinases/genética , Vasos Retinianos/crescimento & desenvolvimento , Animais , Células Endoteliais/citologia , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Proteínas dos Microfilamentos/genética , Fenótipo , Via de Sinalização Wnt/genética
10.
Curr Biol ; 15(24): 2243-8, 2005 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-16271475

RESUMO

Most nerve cells communicate with each other through synaptic transmission at chemical synapses. The regulated exocytosis of neurotransmitters, hormones, and peptides occurs at specialized membrane areas through Ca2+-triggered fusion of secretory vesicles with the plasma membrane . Prior to fusion, vesicles are docked at the plasma membrane and must then be rendered fusion-competent through a process called priming. The molecular mechanism underlying this priming process is most likely the formation of the SNARE complex consisting of Syntaxin 1, SNAP-25, and Synaptobrevin 2. Members of the Munc13 protein family consisting of Munc13-1, -2, -3, and -4 were found to be absolutely required for this priming process . In the present study, we identified the minimal Munc13-1 domain that is responsible for its priming activity. Using Munc13-1 deletion constructs in an electrophysiological gain-of-function assay of chromaffin-granule secretion, we show that priming activity is mediated by the C-terminal residues 1100-1735 of Munc13-1, which contains both Munc13-homology domains and the C-terminal C2 domain. Priming by Munc13-1 appears to require its interaction with Syntaxin 1 because point mutants that do not bind Syntaxin 1 do not prime chromaffin granules.


Assuntos
Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Estrutura Terciária de Proteína , Transmissão Sináptica/genética , Vesículas Sinápticas/metabolismo , Animais , Western Blotting , Cálcio/metabolismo , Células Cromafins , Grânulos Cromafim/metabolismo , Eletrofisiologia , Proteínas de Fluorescência Verde , Camundongos , Transmissão Sináptica/fisiologia , Sintaxina 1/metabolismo
11.
Neuron ; 95(5): 983-985, 2017 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-28858624

RESUMO

CNS angiogenesis and blood-brain barrier integrity are controlled by the canonical Wnt pathway. In this issue of Neuron, Cho et al. (2017) use advanced mouse genetics and biochemical experiments to unravel the ligand-specific association of membrane proteins GPR124 and RECK with Wnt receptor complexes.


Assuntos
Neovascularização Fisiológica , Receptores Acoplados a Proteínas G/química , Animais , Barreira Hematoencefálica , Camundongos , Receptores Wnt , Via de Sinalização Wnt
12.
Nat Commun ; 8: 16050, 2017 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-28675177

RESUMO

Angiogenesis and blood-brain barrier formation are required for normal central nervous system (CNS) function. Both processes are controlled by Wnt or Norrin (NDP) ligands, Frizzled (FZD) receptors, and ß-catenin-dependent signalling in vascular endothelial cells. In the retina, FZD4 and the ligand NDP are critical mediators of signalling and are mutated in familial exudative vitreoretinopathy. Here, we report that NDP is a potent trigger of FZD4 ubiquitination and induces internalization of the NDP receptor complex into the endo-lysosomal compartment. Inhibition of ubiquitinated cargo transport through the multivesicular body (MVB) pathway using a dominant negative ESCRT (endosomal sorting complexes required for transport) component VPS4 EQ strongly impairs NDP/FZD4 signalling in vitro and recapitulates CNS angiogenesis and blood-CNS-barrier defects caused by impaired vascular ß-catenin signalling in mice. These findings provide evidence for an important role of FZD4 endocytosis in NDP/FZD4 signalling and in CNS vascular biology and disease.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Barreira Hematoencefálica/metabolismo , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Células Endoteliais/metabolismo , Proteínas do Olho/metabolismo , Receptores Frizzled/metabolismo , Lisossomos/metabolismo , Neovascularização Fisiológica , Proteínas do Tecido Nervoso/metabolismo , Vasos Retinianos/crescimento & desenvolvimento , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Endossomos/metabolismo , Oftalmopatias Hereditárias/genética , Oftalmopatias Hereditárias/metabolismo , Proteínas do Olho/genética , Vitreorretinopatias Exsudativas Familiares , Receptores Frizzled/genética , Células HEK293 , Células HeLa , Humanos , Técnicas In Vitro , Camundongos , Corpos Multivesiculares/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética , Transporte Proteico , Retina , Doenças Retinianas/genética , Doenças Retinianas/metabolismo , Ubiquitinação , Via de Sinalização Wnt
13.
Cell Rep ; 19(13): 2809-2822, 2017 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-28658627

RESUMO

Accessory proteins in Frizzled (FZD) receptor complexes are thought to determine ligand selectivity and signaling amplitude. Genetic evidence indicates that specific combinations of accessory proteins and ligands mediate vascular ß-catenin signaling in different CNS structures. In the retina, the tetraspanin TSPAN12 and the ligand norrin (NDP) mediate angiogenesis, and both genes are linked to familial exudative vitreoretinopathy (FEVR), yet the molecular function of TSPAN12 remains poorly understood. Here, we report that TSPAN12 is an essential component of the NDP receptor complex and interacts with FZD4 and NDP via its extracellular loops, consistent with an action as co-receptor that enhances FZD4 ligand selectivity for NDP. FEVR-linked mutations in TSPAN12 prevent the incorporation of TSPAN12 into the NDP receptor complex. In vitro and in Xenopus embryos, TSPAN12 alleviates defects of FZD4 M105V, a mutation that destabilizes the NDP/FZD4 interaction. This study sheds light on the poorly understood function of accessory proteins in FZD signaling.


Assuntos
Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Receptores Frizzled/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Tetraspaninas/metabolismo , Receptores Frizzled/genética , Humanos , Mutação de Sentido Incorreto , Transdução de Sinais
14.
Neural Dev ; 11(1): 19, 2016 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-27784329

RESUMO

BACKGROUND: Newborn neurons often migrate before undergoing final differentiation, extending neurites, and forming synaptic connections. Therefore, neuronal migration is crucial for establishing neural circuitry during development. In the developing spinal cord, neuroprogenitors first undergo radial migration within the ventricular zone. Differentiated neurons continue to migrate tangentially before reaching the final positions. The molecular pathways that regulate these migration processes remain largely unknown. Our previous study suggests that the DCC receptor is important for the migration of the dorsal spinal cord progenitors and interneurons. In this study, we determined the involvement of the Netrin1 ligand and the ROBO3 coreceptor in the migration. RESULTS: By pulse labeling neuroprogenitors with electroporation, we examined their radial migration in Netrin1 (Ntn1), Dcc, and Robo3 knockout mice. We found that all three mutants exhibit delayed migration. Furthermore, using immunohistochemistry of the BARHL2 interneuron marker, we found that the mediolateral and dorsoventral migration of differentiated dorsal interneurons is also delayed. Together, our results suggest that Netrin1/DCC signaling induce neuronal migration in the dorsal spinal cord. CONCLUSIONS: Netrin1, DCC, and ROBO3 have been extensively studied for their functions in regulating axon guidance in the spinal commissural interneurons. We reveal that during earlier development of dorsal interneurons including commissural neurons, these molecules play an important role in promoting cell migration.


Assuntos
Movimento Celular , Interneurônios/fisiologia , Fatores de Crescimento Neural/fisiologia , Receptores de Superfície Celular/fisiologia , Medula Espinal/crescimento & desenvolvimento , Proteínas Supressoras de Tumor/fisiologia , Animais , Receptor DCC , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Netrina-1 , Células-Tronco Neurais/fisiologia , Receptores de Superfície Celular/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/genética
15.
Elife ; 52016 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-27223328

RESUMO

RNA-binding proteins (RBPs) control multiple aspects of post-transcriptional gene regulation and function during various biological processes in the nervous system. To further reveal the functional significance of RBPs during neural development, we carried out an in vivo RNAi screen in the dorsal spinal cord interneurons, including the commissural neurons. We found that the NOVA family of RBPs play a key role in neuronal migration, axon outgrowth, and axon guidance. Interestingly, Nova mutants display similar defects as the knockout of the Dcc transmembrane receptor. We show here that Nova deficiency disrupts the alternative splicing of Dcc, and that restoring Dcc splicing in Nova knockouts is able to rescue the defects. Together, our results demonstrate that the production of DCC splice variants controlled by NOVA has a crucial function during many stages of commissural neuron development.


Assuntos
Processamento Alternativo , Antígenos de Neoplasias/metabolismo , Orientação de Axônios , Interneurônios Comissurais/fisiologia , Receptor DCC/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Ligação a RNA/metabolismo , Medula Espinal/embriologia , Animais , Antígenos de Neoplasias/genética , Testes Genéticos , Camundongos , Camundongos Knockout , Antígeno Neuro-Oncológico Ventral , Interferência de RNA , Proteínas de Ligação a RNA/genética
16.
Elife ; 52016 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-27223325

RESUMO

The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.


Assuntos
Antígenos de Neoplasias/metabolismo , Orientação de Axônios , Córtex Cerebral/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/fisiologia , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Animais , Camundongos , Camundongos Knockout , Antígeno Neuro-Oncológico Ventral
17.
PLoS One ; 10(7): e0132013, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26177550

RESUMO

In the retina blood vessels are required to support a high metabolic rate, however, uncontrolled vascular growth can lead to impaired vision and blindness. Subretinal vascularization (SRV), one type of pathological vessel growth, occurs in retinal angiomatous proliferation and proliferative macular telangiectasia. In these diseases SRV originates from blood vessels within the retina. We use mice with a targeted disruption in the Vldl-receptor (Vldlr) gene as a model to study SRV with retinal origin. We find that Vldlr mRNA is strongly expressed in the neuroretina, and we observe both vascular and neuronal phenotypes in Vldlr-/- mice. Unexpectedly, horizontal cell (HC) neurites are mistargeted prior to SRV in this model, and the majority of vascular lesions are associated with mistargeted neurites. In Foxn4-/- mice, which lack HCs and display reduced amacrine cell (AC) numbers, we find severe defects in intraretinal capillary development. However, SRV is not suppressed in Foxn4-/-;Vldlr-/- mice, which reveals that mistargeted HC neurites are not required for vascular lesion formation. In the absence of VLDLR, the intraretinal capillary plexuses form in an inverse order compared to normal development, and subsequent to this early defect, vascular proliferation is increased. We conclude that SRV in the Vldlr-/- model is associated with mistargeted neurites and that SRV is preceded by altered retinal vascular development.


Assuntos
Neuritos/metabolismo , Receptores de LDL/deficiência , Neovascularização Retiniana/patologia , Animais , Animais Recém-Nascidos , Capilares/patologia , Proliferação de Células , Progressão da Doença , Proteínas do Olho/metabolismo , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Receptores de LDL/metabolismo , Retina/metabolismo , Retina/patologia , Neovascularização Retiniana/metabolismo
18.
Cell ; 118(3): 389-401, 2004 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-15294163

RESUMO

The efficacy of synaptic transmission between neurons can be altered transiently during neuronal network activity. This phenomenon of short-term plasticity is a key determinant of network properties; is involved in many physiological processes such as motor control, sound localization, or sensory adaptation; and is critically dependent on cytosolic [Ca2+]. However, the underlying molecular mechanisms and the identity of the Ca2+ sensor/effector complexes involved are unclear. We now identify a conserved calmodulin binding site in UNC-13/Munc13s, which are essential regulators of synaptic vesicle priming and synaptic efficacy. Ca2+ sensor/effector complexes consisting of calmodulin and Munc13s regulate synaptic vesicle priming and synaptic efficacy in response to a residual [Ca2+] signal and thus shape short-term plasticity characteristics during periods of sustained synaptic activity.


Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Sítios de Ligação , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas do Tecido Nervoso/genética
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