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1.
Hum Mol Genet ; 24(13): 3775-91, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25859007

RESUMO

Distinct mutations in the centrosomal-cilia protein CEP290 lead to diverse clinical findings in syndromic ciliopathies. We show that CEP290 localizes to the transition zone in ciliated cells, precisely to the region of Y-linkers between central microtubules and plasma membrane. To create models of CEP290-associated ciliopathy syndromes, we generated Cep290(ko/ko) and Cep290(gt/gt) mice that produce no or a truncated CEP290 protein, respectively. Cep290(ko/ko) mice exhibit early vision loss and die from hydrocephalus. Retinal photoreceptors in Cep290(ko/ko) mice lack connecting cilia, and ciliated ventricular ependyma fails to mature. The minority of Cep290(ko/ko) mice that escape hydrocephalus demonstrate progressive kidney pathology. Cep290(gt/gt) mice die at mid-gestation, and the occasional Cep290(gt/gt) mouse that survives shows hydrocephalus and severely cystic kidneys. Partial loss of CEP290-interacting ciliopathy protein MKKS mitigates lethality and renal pathology in Cep290(gt/gt) mice. Our studies demonstrate domain-specific functions of CEP290 and provide novel therapeutic paradigms for ciliopathies.


Assuntos
Cílios/metabolismo , Hidrocefalia/genética , Doenças Renais Císticas/genética , Proteínas Nucleares/genética , Animais , Cílios/genética , Modelos Animais de Doenças , Feminino , Humanos , Hidrocefalia/metabolismo , Doenças Renais Císticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/metabolismo , Especificidade de Órgãos
2.
Invest Ophthalmol Vis Sci ; 55(9): 6031-40, 2014 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-25159211

RESUMO

PURPOSE: The aryl hydrocarbon receptor (AHR) is a ligand-activated nuclear receptor that regulates cellular response to environmental signals, including UV and blue wavelength light. This study was undertaken to elucidate AHR function in retinal homeostasis. METHODS: RNA-seq data sets were examined for Ahr expression in the mouse retina and rod photoreceptors. The Ahr(-/-) mice were evaluated by fundus imaging, optical coherence tomography, histology, immunohistochemistry, and ERG. For light damage experiments, adult mice were exposed to 14,000 to 15,000 lux of diffuse white light for 2 hours. RESULTS: In mouse retina, Ahr transcripts were upregulated during development, with continued increase in aging rod photoreceptors. Fundus examination of 3-month-old Ahr(-/-) mice revealed subretinal autofluorescent spots, which increased in number with age and following acute light exposure. Ahr(-/-) retina also showed subretinal microglia accumulation that correlated with autofluorescence changes, RPE abnormalities, and reactivity against immunoglobulin, complement factor H, and glial fibrillary acidic protein. Functionally, Ahr(-/-) mice displayed reduced ERG c-wave amplitudes. CONCLUSIONS: The Ahr(-/-) mice exhibited subretinal accumulation of microglia and focal RPE atrophy, phenotypes observed in AMD. Together with a recently published report on another Ahr(-/-) mouse model, our study suggests that AHR has a protective role in the retina as an environmental stress sensor. As such, its altered function may contribute to human AMD progression and provide a target for pharmacological intervention.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Microglia/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Degeneração Retiniana/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Atrofia/metabolismo , Atrofia/patologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Modelos Animais de Doenças , Fundo de Olho , Deleção de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/patologia , Receptores de Hidrocarboneto Arílico/imunologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinite/metabolismo , Retinite/patologia , Tomografia de Coerência Óptica , Transcriptoma
3.
Nat Commun ; 5: 4207, 2014 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-24947469

RESUMO

The primary cilium originates from the mother centriole and participates in critical functions during organogenesis. Defects in cilia biogenesis or function lead to pleiotropic phenotypes. Mutations in centrosome-cilia gene CC2D2A result in Meckel and Joubert syndromes. Here we generate a Cc2d2a(-/-) mouse that recapitulates features of Meckel syndrome including embryonic lethality and multiorgan defects. Cilia are absent in Cc2d2a(-/-) embryonic node and other somatic tissues; disruption of cilia-dependent Shh signalling appears to underlie exencephaly in mutant embryos. The Cc2d2a(-/-) mouse embryonic fibroblasts (MEFs) lack cilia, although mother centrioles and pericentriolar proteins are detected. Odf2, associated with subdistal appendages, is absent and ninein is reduced in mutant MEFs. In Cc2d2a(-/-) MEFs, subdistal appendages are lacking or abnormal by transmission electron microscopy. Consistent with this, CC2D2A localizes to subdistal appendages by immuno-EM in wild-type cells. We conclude that CC2D2A is essential for the assembly of subdistal appendages, which anchor cytoplasmic microtubules and prime the mother centriole for axoneme biogenesis.


Assuntos
Centríolos/metabolismo , Cílios/patologia , Proteínas/genética , Alelos , Animais , Transporte Biológico , Centrossomo/ultraestrutura , Cílios/genética , Citoplasma/metabolismo , Proteínas do Citoesqueleto , Fibroblastos/metabolismo , Citometria de Fluxo , Proteínas Hedgehog/metabolismo , Macaca mulatta , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Microtúbulos/metabolismo , Mutação , Fenótipo , Proteínas/fisiologia , Transdução de Sinais , Transgenes
4.
Dev Biol ; 384(1): 41-52, 2013 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-24095903

RESUMO

The integrity and function of epithelial tissues depend on the establishment and maintenance of defining characteristics of epithelial cells, cell-cell adhesion and cell polarity. Disruption of these characteristics can lead to the loss of epithelial identity through a process called epithelial to mesenchymal transition (EMT), which can contribute to pathological conditions such as tissue fibrosis and invasive cancer. In invertebrates, the epithelial polarity gene scrib plays a critical role in establishing and maintaining cell adhesion and polarity. In this study we asked if the mouse homolog, Scrib, is required for establishment and/or maintenance of epithelial identity in vivo. To do so, we conditionally deleted Scrib in the head ectoderm tissue that gives rise to both the ocular lens and the corneal epithelium. Deletion of Scrib in the lens resulted in a change in epithelial cell shape from cuboidal to flattened and elongated. Early in the process, the cell adhesion protein, E-cadherin, and apical polarity protein, ZO-1, were downregulated and the myofibroblast protein, αSMA, was upregulated, suggesting EMT was occurring in the Scrib deficient lenses. Correlating temporally with the upregulation of αSMA, Smad3 and Smad4, TGFß signaling intermediates, accumulated in the nucleus and Snail, a TGFß target and transcriptional repressor of the gene encoding E-cadherin, was upregulated. Pax6, a lens epithelial transcription factor required to maintain lens epithelial cell identity also was downregulated. Loss of Scrib in the corneal epithelium also led to molecular changes consistent with EMT, suggesting that the effect of Scrib deficiency was not unique to the lens. Together, these data indicate that mammalian Scrib is required to maintain epithelial identity and that loss of Scrib can culminate in EMT, mediated, at least in part, through TGFß signaling.


Assuntos
Células Epiteliais/citologia , Transição Epitelial-Mesenquimal/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Animais , Caderinas/genética , Caderinas/metabolismo , Adesão Celular , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cristalino/metabolismo , Camundongos , Camundongos Transgênicos , Fator de Crescimento Transformador beta/metabolismo
5.
Development ; 140(6): 1330-41, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23406904

RESUMO

Dysfunction or death of photoreceptors is the primary cause of vision loss in retinal and macular degenerative diseases. As photoreceptors have an intimate relationship with the retinal pigment epithelium (RPE) for exchange of macromolecules, removal of shed membrane discs and retinoid recycling, an improved understanding of the development of the photoreceptor-RPE complex will allow better design of gene- and cell-based therapies. To explore the epigenetic contribution to retinal development we generated conditional knockout alleles of DNA methyltransferase 1 (Dnmt1) in mice. Conditional Dnmt1 knockdown in early eye development mediated by Rx-Cre did not produce lamination or cell fate defects, except in cones; however, the photoreceptors completely lacked outer segments despite near normal expression of phototransduction and cilia genes. We also identified disruption of RPE morphology and polarization as early as E15.5. Defects in outer segment biogenesis were evident with Dnmt1 exon excision only in RPE, but not when excision was directed exclusively to photoreceptors. We detected a reduction in DNA methylation of LINE1 elements (a measure of global DNA methylation) in developing mutant RPE as compared with neural retina, and of Tuba3a, which exhibited dramatically increased expression in mutant retina. These results demonstrate a unique function of DNMT1-mediated DNA methylation in controlling RPE apicobasal polarity and neural retina differentiation. We also establish a model to study the epigenetic mechanisms and signaling pathways that guide the modulation of photoreceptor outer segment morphogenesis by RPE during retinal development and disease.


Assuntos
Permeabilidade da Membrana Celular/fisiologia , DNA (Citosina-5-)-Metiltransferases/genética , Morfogênese/genética , Segmento Externo das Células Fotorreceptoras da Retina/fisiologia , Epitélio Pigmentado da Retina/fisiologia , Animais , Permeabilidade da Membrana Celular/genética , Polaridade Celular/genética , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA/genética , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Transgênicos , Análise em Microsséries , Morfogênese/fisiologia , Especificidade de Órgãos/genética , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Epitélio Pigmentado da Retina/embriologia , Epitélio Pigmentado da Retina/crescimento & desenvolvimento , Epitélio Pigmentado da Retina/metabolismo , Transcriptoma
6.
PLoS One ; 7(9): e42446, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22984402

RESUMO

Humans with Hermansky-Pudlak Syndrome (HPS) or ocular albinism (OA1) display abnormal aspects of organelle biogenesis. The multigenic disorder HPS displays broad defects in biogenesis of lysosome-related organelles including melanosomes, platelet dense granules, and lysosomes. A phenotype of ocular pigmentation in OA1 is a smaller number of macromelanosomes, in contrast to HPS, where in many cases the melanosomes are smaller than normal. In these studies we define the role of the Mreg(dsu) gene, which suppresses the coat color dilution of Myo5a, melanophilin, and Rab27a mutant mice in maintaining melanosome size and distribution. We show that the product of the Mreg(dsu) locus, melanoregulin (MREG), interacts both with members of the HPS BLOC-2 complex and with Oa1 in regulating melanosome size. Loss of MREG function facilitates increase in the size of micromelanosomes in the choroid of the HPS BLOC-2 mutants ruby, ruby2, and cocoa, while a transgenic mouse overexpressing melanoregulin corrects the size of retinal pigment epithelium (RPE) macromelanosomes in Oa1(ko/ko) mice. Collectively, these results suggest that MREG levels regulate pigment incorporation into melanosomes. Immunohistochemical analysis localizes melanoregulin not to melanosomes, but to small vesicles in the cytoplasm of the RPE, consistent with a role for this protein in regulating membrane interactions during melanosome biogenesis. These results provide the first link between the BLOC pathway and Oa1 in melanosome biogenesis, thus supporting the hypothesis that intracellular G-protein coupled receptors may be involved in the biogenesis of other organelles. Furthermore these studies provide the foundation for therapeutic approaches to correct the pigment defects in the RPE of HPS and OA1.


Assuntos
Albinismo Ocular/genética , Proteínas de Transporte/metabolismo , Loci Gênicos/genética , Organelas/metabolismo , Albinismo Ocular/patologia , Animais , Proteínas de Transporte/genética , Linhagem Celular , Corioide/metabolismo , Corioide/patologia , Corioide/ultraestrutura , Dosagem de Genes/genética , Síndrome de Hermanski-Pudlak/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Melanossomas/metabolismo , Melanossomas/patologia , Melanossomas/ultraestrutura , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Mutação/genética , Tamanho das Organelas , Epitélio Pigmentado Ocular/metabolismo , Epitélio Pigmentado Ocular/patologia , Epitélio Pigmentado Ocular/ultraestrutura , Transporte Proteico , Proteínas de Transporte Vesicular
7.
J Clin Invest ; 122(4): 1233-45, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22446187

RESUMO

Cilia are highly specialized microtubule-based organelles that have pivotal roles in numerous biological processes, including transducing sensory signals. Defects in cilia biogenesis and transport cause pleiotropic human ciliopathies. Mutations in over 30 different genes can lead to cilia defects, and complex interactions exist among ciliopathy-associated proteins. Mutations of the centrosomal protein 290 kDa (CEP290) lead to distinct clinical manifestations, including Leber congenital amaurosis (LCA), a hereditary cause of blindness due to photoreceptor degeneration. Mice homozygous for a mutant Cep290 allele (Cep290rd16 mice) exhibit LCA-like early-onset retinal degeneration that is caused by an in-frame deletion in the CEP290 protein. Here, we show that the domain deleted in the protein encoded by the Cep290rd16 allele directly interacts with another ciliopathy protein, MKKS. MKKS mutations identified in patients with the ciliopathy Bardet-Biedl syndrome disrupted this interaction. In zebrafish embryos, combined subminimal knockdown of mkks and cep290 produced sensory defects in the eye and inner ear. Intriguingly, combinations of Cep290rd16 and Mkksko alleles in mice led to improved ciliogenesis and sensory functions compared with those of either mutant alone. We propose that altered association of CEP290 and MKKS affects the integrity of multiprotein complexes at the cilia transition zone and basal body. Amelioration of the sensory phenotypes caused by specific mutations in one protein by removal of an interacting domain/protein suggests a possible novel approach for treating human ciliopathies.


Assuntos
Antígenos de Neoplasias/genética , Síndrome de Bardet-Biedl/genética , Cílios/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Chaperoninas do Grupo II/genética , Amaurose Congênita de Leber/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Transtornos das Sensações/genética , Alelos , Sequência de Aminoácidos , Animais , Chaperoninas/deficiência , Chaperoninas/genética , Chaperoninas/fisiologia , Análise Mutacional de DNA , Orelha/anormalidades , Orelha/embriologia , Anormalidades do Olho/embriologia , Anormalidades do Olho/genética , Teste de Complementação Genética , Chaperoninas do Grupo II/deficiência , Chaperoninas do Grupo II/fisiologia , Células HEK293 , Células Ciliadas Auditivas/ultraestrutura , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/fisiologia , Dados de Sequência Molecular , Proteínas Nucleares/deficiência , Proteínas Nucleares/fisiologia , Neurônios Receptores Olfatórios/ultraestrutura , Cílio Conector dos Fotorreceptores/ultraestrutura , Mapeamento de Interação de Proteínas , Transtornos das Sensações/patologia , Transtornos das Sensações/prevenção & controle , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia
8.
J Neurosci ; 32(2): 528-41, 2012 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-22238088

RESUMO

Cone photoreceptors are the primary initiator of visual transduction in the human retina. Dysfunction or death of rod photoreceptors precedes cone loss in many retinal and macular degenerative diseases, suggesting a rod-dependent trophic support for cone survival. Rod differentiation and homeostasis are dependent on the basic motif leucine zipper transcription factor neural retina leucine zipper (NRL). The loss of Nrl (Nrl(-/-)) in mice results in a retina with predominantly S-opsin-containing cones that exhibit molecular and functional characteristics of wild-type cones. Here, we report that Nrl(-/-) retina undergoes a rapid but transient period of degeneration in early adulthood, with cone apoptosis, retinal detachment, alterations in retinal vessel structure, and activation and translocation of retinal microglia. However, cone degeneration stabilizes by 4 months of age, resulting in a thinner but intact outer nuclear layer with residual cones expressing S- and M-opsins and a preserved photopic electroretinogram. At this stage, microglia translocate back to the inner retina and reacquire a quiescent morphology. Gene profiling analysis during the period of transient degeneration reveals misregulation of genes related to stress response and inflammation, implying their involvement in cone death. The Nrl(-/-) mouse illustrates the long-term viability of cones in the absence of rods and retinal pigment epithelium defects in a rodless retina. We propose that Nrl(-/-) retina may serve as a model for elucidating mechanisms of cone homeostasis and degeneration that would be relevant to understanding diseases of the cone-dominant human macula.


Assuntos
Apoptose/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas do Olho/genética , Retina/anormalidades , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/fisiopatologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/deficiência , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Descolamento Retiniano/genética , Descolamento Retiniano/patologia , Descolamento Retiniano/fisiopatologia
9.
Cilia ; 1(1): 22, 2012 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-23351659

RESUMO

Ciliopathies encompass a broad array of clinical findings associated with genetic defects in biogenesis and/or function of the primary cilium, a ubiquitous organelle involved in the transduction of diverse biological signals. Degeneration or dysfunction of retinal photoreceptors is frequently observed in diverse ciliopathies. The sensory cilium in a photoreceptor elaborates into unique outer segment discs that provide extensive surface area for maximal photon capture and efficient visual transduction. The daily renewal of approximately 10% of outer segments requires a precise control of ciliary transport. Here, we review the ciliopathies with associated retinal degeneration, describe the distinctive structure of the photoreceptor cilium, and discuss mouse models that allow investigations into molecular mechanisms of cilia biogenesis and defects. We have specifically focused on two ciliary proteins - CEP290 and RPGR - that underlie photoreceptor degeneration and syndromic ciliopathies. Mouse models of CEP290 and RPGR disease, and of their multiple interacting partners, have helped unravel new functional insights into cell type-specific phenotypic defects in distinct ciliary proteins. Elucidation of multifaceted ciliary functions and associated protein complexes will require concerted efforts to assimilate diverse datasets from in vivo and in vitro studies. We therefore discuss a possible framework for investigating genetic networks associated with photoreceptor cilia biogenesis and pathology.

10.
J Biol Chem ; 286(32): 28276-86, 2011 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-21685394

RESUMO

Primary cilia regulate polarized protein trafficking in photoreceptors, which are dynamic and highly compartmentalized sensory neurons of retina. The ciliary protein Cep290 modulates cilia formation and is frequently mutated in syndromic and non-syndromic photoreceptor degeneration. However, the underlying mechanism of associated retinopathy is unclear. Using the Cep290 mutant mouse rd16 (retinal degeneration 16), we show that Cep290-mediated photoreceptor degeneration is associated with aberrant accumulation of its novel interacting partner Rkip (Raf-1 kinase inhibitory protein). This effect is phenocopied by morpholino-mediated depletion of cep290 in zebrafish. We further demonstrate that ectopic accumulation of Rkip leads to defective cilia formation in zebrafish and cultured cells, an effect mediated by its interaction with the ciliary GTPase Rab8A. Our data suggest that Rkip prevents cilia formation and is associated with Cep290-mediated photoreceptor degeneration. Furthermore, our results indicate that preventing accumulation of Rkip could potentially ameliorate such degeneration.


Assuntos
Antígenos de Neoplasias/metabolismo , Transtornos da Motilidade Ciliar/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Degeneração Retiniana/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Antígenos de Neoplasias/genética , Chlorocebus aethiops , Cílios/genética , Cílios/metabolismo , Cílios/patologia , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/patologia , Células HEK293 , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteína de Ligação a Fosfatidiletanolamina/genética , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
11.
Hum Mol Genet ; 20(7): 1411-23, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21245082

RESUMO

Leber congenital amaurosis (LCA), a severe autosomal recessive childhood blindness, is caused by mutations in at least 15 genes. The most common molecular form is a ciliopathy due to NPHP6 (CEP290) mutations and subjects have profound loss of vision. A similarly severe phenotype occurs in the related ciliopathy NPHP5 (IQCB1)-LCA. Recent success of retinal gene therapy in one form of LCA prompted the question whether we know enough about human NPHP5 and NPHP6 disease to plan such treatment. We determined that there was early-onset rapid degeneration of rod photoreceptors in young subjects with these ciliopathies. Rod outer segment (OS) lamination, when detectable, was disorganized. Retinal pigment epithelium lipofuscin accumulation indicated that rods had existed in the past in most subjects. In contrast to early rod losses, the all-cone human fovea in NPHP5- and NPHP6-LCA of all ages retained cone nuclei, albeit with abnormal inner segments and OS. The rd16 mouse, carrying a hypomorphic Nphp6 allele, was a good model of the rod-dominant human extra-foveal retina. Rd16 mice showed normal genesis of photoreceptors, including the formation of cilia, followed by abnormal elaboration of OS and rapid degeneration. To produce a model of the all-cone human fovea in NPHP6-LCA, we generated rd16;Nrl-/- double-mutant mice. They showed substantially retained cone photoreceptors with disproportionate cone function loss, such as in the human disease. NPHP5- and NPHP6-LCA across a wide age spectrum are thus excellent candidates for cone-directed gene augmentation therapy, and the rd16;Nrl-/- mouse is an appropriate model for pre-clinical proof-of-concept studies.


Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Terapia Genética , Amaurose Congênita de Leber/terapia , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Adolescente , Adulto , Alelos , Animais , Antígenos de Neoplasias/genética , Proteínas de Ligação a Calmodulina/genética , Criança , Cílios , Feminino , Humanos , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/metabolismo , Masculino , Camundongos , Camundongos Mutantes , Pessoa de Meia-Idade , Mutação , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Células Fotorreceptoras Retinianas Cones/patologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Segmento Externo da Célula Bastonete/metabolismo , Segmento Externo da Célula Bastonete/patologia
12.
J Biol Chem ; 284(16): 10877-89, 2009 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-19240024

RESUMO

Melanoregulin (MREG), the product of the Mreg(dsu) gene, is a small highly charged protein, hypothesized to play a role in organelle biogenesis due to its effect on pigmentation in dilute, ashen, and leaden mutant mice. Here we provide evidence that MREG is required in lysosome-dependent phagosome degradation. In the Mreg(-/-) mouse, we show that loss of MREG function results in phagosome accumulation due to delayed degradation of engulfed material. Over time, the Mreg(-/-) mouse retinal pigment epithelial cells accumulate the lipofuscin component, A2E. MREG-deficient human and mouse retinal pigment epithelial cells exhibit diminished activity of the lysosomal hydrolase, cathepsin D, due to defective processing. Moreover, MREG localizes to small intracellular vesicles and associates with the endosomal phosphoinositide, phosphatidylinositol 3,5-biphosphate. Collectively, these studies suggest that MREG is required for lysosome maturation and support a role for MREG in intracellular trafficking.


Assuntos
Proteínas de Transporte/metabolismo , Células Epiteliais/metabolismo , Lisossomos/metabolismo , Epitélio Pigmentado Ocular/citologia , Animais , Proteínas de Transporte/genética , Catepsina D/metabolismo , Linhagem Celular , Células Epiteliais/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lipofuscina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose/fisiologia , Fosfatidiletanolaminas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Compostos de Piridínio/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Retinaldeído/análogos & derivados , Retinaldeído/metabolismo , Retinoides/metabolismo
13.
Biochemistry ; 46(5): 1256-72, 2007 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-17260955

RESUMO

Peripherin-2, the product of the rds gene, is a tetraspanin protein. In this study, we show that peripherin-2 forms a complex with melanoregulin (MREG), the product of the Mreg locus. Genetic studies suggest that MREG is involved in organelle biogenesis. In this study, we explore the role of this protein in processes associated with the formation of disk membranes, specialized organelles of photoreceptor rod cells. MREG antibodies were generated and found to be immunoreactive with a 28 kDa protein in retinal extracts, bovine OS, ARPE-19 cells, and rat RPE. MREG colocalized with peripherin-2 in WT (CB6F1/J) and in rds+/- retinas. Western blots of serial tangential sections confirmed the close association of these two proteins within the IS and basal outer segment of rods. Immunoprecipitation (IP) of OS extracts showed formation of a complex between MREG and peripherin-2-ROM-1 hetero-oligomers. This interaction was confirmed with pulldown analyses in which the GST-PerCter protein selectively pulled down His-MREG and His-MREG selectively pulled down PerCter. Biacore analysis using peptide inhibitors and per-2 truncation mutant studies allowed us to map the MREG binding site on per-2 to the last five residues of the C-terminus (Gln341-Gly346), and kinetic data predicted a KD of 80 nM for PerCter-MREG binding. Finally, the effect of MREG on photoreceptor specific membrane fusion was assayed using a disk-plasma membrane cell free assay. Preincubation of target membranes with MREG resulted in a dose-dependent inhibition of fusion with an IC50 in the submicromolar range. Collectively, these results suggest that this newly identified protein regulates peripherin-2 function.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Sítios de Ligação , Proteínas de Transporte/fisiologia , Bovinos , Linhagem Celular , Membrana Celular , Humanos , Proteínas de Filamentos Intermediários/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Fusão de Membrana , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/fisiologia , Disco Óptico/ultraestrutura , Periferinas , Células Fotorreceptoras/ultraestrutura , Ratos , Retina/química , Retina/citologia
14.
Mol Cell Biol ; 26(18): 6913-22, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16943432

RESUMO

Zinc finger protein 423 (also known as Ebf-associated zinc finger protein, Ebfaz) binds to and negatively regulates Ebf1, a basic helix-loop-helix transcription factor required for B-cell lineage commitment and olfactory epithelium development. Zfp423 also binds to Smad1/Smad4 in response to Bmp2 signaling. Zfp423 contains 30 Krüppel-like zinc fingers that are organized into discrete clusters; some zinc fingers are used to bind DNA, while others mediate Zfp423's interaction with other signaling proteins such as Ebf1 and Smad1/Smad4. Previously, we showed that Zfp423 is an oncogene whose upregulation following retroviral integration in murine B cells leads to an arrest in B-cell differentiation and the subsequent development of B-cell lymphomas. To study the biological functions of Zfp423 in vivo, we used recombineering and gene targeting to generate mice that carry conditional as well as null alleles of Zfp423. Homozygous Zfp423 null mice are runted and ataxic, the cerebellum is underdeveloped, and the vermis is severely reduced. In the remaining cerebellar structures, the Purkinje cells are poorly developed and mislocalized. In mice carrying a hypomorphic Zfp423 gene trap allele, lacZ expression in the cerebellum correlates with the Purkinje cell layer, suggesting that these phenotypes are a result of a Purkinje cell-intrinsic defect.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células de Purkinje/citologia , Fatores de Transcrição/metabolismo , Alelos , Animais , Animais Recém-Nascidos , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Éxons/genética , Feminino , Regulação da Expressão Gênica , Genoma/genética , Íntrons/genética , Masculino , Camundongos , Camundongos Knockout , Tamanho do Órgão , Células de Purkinje/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
15.
J Neurosci ; 26(19): 5265-75, 2006 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-16687519

RESUMO

Planar cell polarity (PCP) is a process in which cells develop with uniform orientation within the plane of an epithelium. To begin to elucidate the mechanisms of PCP in vertebrates, the localization of the protein Vangl2 (Van Gogh-like) was determined during the development of the mammalian cochlea. Results indicate that Vangl2 becomes asymmetrically localized to specific cell-cell boundaries along the axis of polarization and that this asymmetry is lost in PCP mutants. In addition, PDZ2 (postsynaptic density/Discs large/zona occludens 1), PDZ3, and PDZ4 of the PCP protein Scrb1 (Scribble) are shown to bind to the C-terminal PDZ binding domain of Vangl2, suggesting that Scrb1 plays a direct role in asymmetric targeting of Vangl2. Finally, Fz3 (Frizzled), a newly demonstrated mediator of PCP, is also asymmetrically localized in a pattern that matches that of Vangl2. The presence and asymmetry of Fz3 at the membrane is shown to be dependent on Vangl2. This result suggests a role for Vangl2 in the targeting or anchoring of Fz3, a hypothesis strengthened by the existence of a physical interaction between the two proteins. Together, our data support the idea that protein asymmetry plays an important role in the development of PCP, but the colocalization and interaction of Fz3 and Vangl2 suggests that novel PCP mechanisms exist in vertebrates.


Assuntos
Cóclea/citologia , Cóclea/metabolismo , Receptores Frizzled/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Polaridade Celular/fisiologia , Células Cultivadas , Camundongos , Distribuição Tecidual
16.
Invest Ophthalmol Vis Sci ; 46(10): 3515-20, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16186328

RESUMO

PURPOSE: To establish a transgenic mouse line that expresses Cre-recombinase in retinal rod bipolar cells for the generation of rod bipolar cell-specific knockout mutants. METHODS: The IRES-Cre-cDNA fragment was inserted into a 173-kb bacterial artificial chromosome (BAC) carrying the intact Pcp2 gene, by using red-mediated recombineering. Transgenic mice were generated with the modified BAC and identified. The Cre-transgenic mice were crossed with ROSA26 and Z/EG reporter mice to detect Cre-recombinase activity. RESULTS: X-gal staining showed that strong Cre-recombinase activities were present in retinal inner nuclear layers and cerebellar Purkinje cells. Double staining with an anti-GFP antibody and an anti-PKCalpha antibody (specific for retinal rod bipolar cells) revealed that Cre-recombinase activity localized exclusively to the rod bipolar cells in the retina. CONCLUSIONS: A mouse BAC-Pcp2-IRES-Cre transgenic line that expresses Cre-recombinase in retinal rod bipolar neurons has been established. Because mutations in some ubiquitously expressed genes may result in retinal degenerative diseases, the mouse strain BAC-Pcp2-IRES-Cre will be a useful new tool for investigating the effects of retinal rod bipolar cell-specific gene inactivation.


Assuntos
Integrases/metabolismo , Interneurônios/enzimologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Animais , Cromossomos Artificiais Bacterianos , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Galactosídeos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Indóis/metabolismo , Integrases/genética , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neuropeptídeos/genética , Gravidez , Células de Purkinje/enzimologia , Células Fotorreceptoras Retinianas Bastonetes/embriologia , beta-Galactosidase/metabolismo
17.
Proc Natl Acad Sci U S A ; 101(48): 16831-6, 2004 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-15550542

RESUMO

MYO5A is a major actin-based vesicle transport motor that binds to one of its cargos, the melanosome, by means of a RAB27A/MLPH receptor. When one of the members of this receptor-motor complex is mutated, the melanosomes clump in the perinuclear region of the melanocyte and are transferred unevenly to the developing hair, leading to a dilution of coat color. Mutation of a fourth gene, dilute suppressor (dsu), suppresses this coat color dilution. MYO5A is required for the peripheral accumulation of melanosomes in melanocytes, but its role in melanosome transfer to neighboring keratinocytes and the hair is unknown. Here, we show that MYO5A is nonessential for melanosome transfer, although pigment incorporation into the hair in MYO5A-deficient mice is uneven, probably due to the clumping of melanosomes that occurs in the perinuclear region of mutant melanocytes. We also show that dsu is caused by a loss-of-function mutation in a unique vertebrate-specific protein that appears to function in an MYO5A-independent pathway to alter pigment incorporation into the hair. Therefore, dsu identifies a unique protein involved in pigmentation of the mammalian hair.


Assuntos
Cor de Cabelo/genética , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Animais , Western Blotting , Cromossomos Bacterianos , Teste de Complementação Genética , Camundongos , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética
18.
EMBO J ; 23(2): 450-9, 2004 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-14713950

RESUMO

Meis1 and Hoxa9 expression is upregulated by retroviral integration in murine myeloid leukemias and in human leukemias carrying MLL translocations. Both genes also cooperate to induce leukemia in a mouse leukemia acceleration assay, which can be explained, in part, by their physical interaction with each other as well as the PBX family of homeodomain proteins. Here we show that Meis1-deficient embryos have partially duplicated retinas and smaller lenses than normal. They also fail to produce megakaryocytes, display extensive hemorrhaging, and die by embryonic day 14.5. In addition, Meis1-deficient embryos lack well-formed capillaries, although larger blood vessels are normal. Definitive myeloerythroid lineages are present in the mutant embryos, but the total numbers of colony-forming cells are dramatically reduced. Mutant fetal liver cells also fail to radioprotect lethally irradiated animals and they compete poorly in repopulation assays even though they can repopulate all hematopoietic lineages. These and other studies showing that Meis1 is expressed at high levels in hematopoietic stem cells (HSCs) suggest that Meis1 may also be required for the proliferation/self-renewal of the HSC.


Assuntos
Anormalidades do Olho/etiologia , Hematopoese , Proteínas de Homeodomínio/fisiologia , Proteínas de Neoplasias/fisiologia , Neovascularização Patológica/etiologia , Animais , Transplante de Células , Embrião de Mamíferos/irrigação sanguínea , Embrião de Mamíferos/citologia , Células Precursoras Eritroides/citologia , Feminino , Feto/citologia , Marcação de Genes , Mutação em Linhagem Germinativa , Hemorragia/etiologia , Proteínas de Homeodomínio/genética , Fígado/citologia , Fígado/embriologia , Megacariócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Meis1 , Células Progenitoras Mieloides/citologia , Proteínas de Neoplasias/genética
19.
Cell ; 114(5): 545-57, 2003 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-13678579

RESUMO

During CNS development, combinatorial expression of transcription factors controls neuronal subtype identity and subsequent axonal trajectory. Regulatory genes designating the routing of retinal ganglion cell (RGC) axons at the optic chiasm to the appropriate hemisphere, a pattern critical for proper binocular vision, have not been identified. Here, we show that the zinc finger transcription factor Zic2, a vertebrate homolog of the Drosophila gene odd-paired, is expressed in RGCs with an uncrossed trajectory during the period when this subpopulation grows from the ventrotemporal retina toward the optic chiasm. Loss- and gain-of-function analyses indicate that Zic2 is necessary and sufficient to regulate RGC axon repulsion by cues at the optic chiasm midline. Moreover, Zic2 expression reflects the extent of binocularity in different species, suggesting that Zic2 is an evolutionarily conserved determinant of RGCs that project ipsilaterally. These data provide evidence for transcriptional coding of axon pathfinding at the midline.


Assuntos
Retina/fisiologia , Fatores de Transcrição/fisiologia , Visão Ocular/fisiologia , Animais , Axônios/metabolismo , Encéfalo/embriologia , Bromodesoxiuridina/farmacologia , Linhagem Celular , Regulação para Baixo , Proteínas de Drosophila/metabolismo , Corantes Fluorescentes/farmacologia , Genótipo , Proteínas de Fluorescência Verde , Proteínas de Homeodomínio/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Mitose , Modelos Biológicos , Proteínas Nucleares , Nervo Óptico/metabolismo , Retina/anatomia & histologia , Retina/embriologia , Retina/metabolismo , Vírus Sindbis/genética , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transcrição Genética , Transfecção
20.
Nature ; 423(6936): 173-7, 2003 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-12724779

RESUMO

In mammals, an example of planar cell polarity (PCP) is the uniform orientation of the hair cell stereociliary bundles within the cochlea. The PCP pathway of Drosophila refers to a conserved signalling pathway that regulates the coordinated orientation of cells or structures within the plane of an epithelium. Here we show that a mutation in Vangl2, a mammalian homologue of the Drosophila PCP gene Strabismus/Van Gogh, results in significant disruptions in the polarization of stereociliary bundles in mouse cochlea as a result of defects in the direction of movement and/or anchoring of the kinocilium within each hair cell. Similar, but less severe, defects are observed in animals containing a mutation in the LAP protein family gene Scrb1 (homologous with Drosophila scribble). Polarization defects in animals heterozygous for Vangl2 and Scrb1 are comparable with Vangl2 homozygotes, demonstrating genetic interactions between these genes in the regulation of PCP in mammals. These results demonstrate a role for the PCP pathway in planar polarization in mammals, and identify Scrb1 as a PCP gene.


Assuntos
Polaridade Celular , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Genótipo , Células Ciliadas Auditivas/anormalidades , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Estrutura Terciária de Proteína , Osso Temporal/anormalidades
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