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1.
J Cell Sci ; 136(20)2023 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-37732459

RESUMO

A characteristic of normal aging and age-related diseases is the remodeling of the cellular organization of a tissue through polyploid cell growth. Polyploidy arises from an increase in nuclear ploidy or the number of nuclei per cell. However, it is not known whether age-induced polyploidy is an adaption to stressors or a precursor to degeneration. Here, we find that abdominal epithelium of the adult fruit fly becomes polyploid with age through generation of multinucleated cells by cell fusion. Inhibition of fusion does not improve the lifespan of the fly, but does enhance its biomechanical fitness, a measure of the healthspan of the animal. Remarkably, Drosophila can maintain their epithelial tension and abdominal movements with age when cell fusion is inhibited. Epithelial cell fusion also appears to be dependent on a mechanical cue, as knockdown of Rho kinase, E-cadherin or α-catenin is sufficient to induce multinucleation in young animals. Interestingly, mutations in α-catenin in mice result in retina pigment epithelial multinucleation associated with macular disease. Therefore, we have discovered that polyploid cells arise by cell fusion and contribute to the decline in the biomechanical fitness of the animal with age.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Camundongos , Drosophila/genética , alfa Catenina , Fusão Celular , Proteínas de Drosophila/genética , Poliploidia
2.
PLoS Genet ; 18(6): e1009798, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35675330

RESUMO

Mutations in the apicobasal polarity gene CRB1 lead to diverse retinal diseases, such as Leber congenital amaurosis, cone-rod dystrophy, retinitis pigmentosa (with and without Coats-like vasculopathy), foveal retinoschisis, macular dystrophy, and pigmented paravenous chorioretinal atrophy. Limited correlation between disease phenotypes and CRB1 alleles, and evidence that patients sharing the same alleles often present with different disease features, suggest that genetic modifiers contribute to clinical variation. Similarly, the retinal phenotype of mice bearing the Crb1 retinal degeneration 8 (rd8) allele varies with genetic background. Here, we initiated a sensitized chemical mutagenesis screen in B6.Cg-Crb1rd8/Pjn, a strain with a mild clinical presentation, to identify genetic modifiers that cause a more severe disease phenotype. Two models from this screen, Tvrm266 and Tvrm323, exhibited increased retinal dysplasia. Genetic mapping with high-throughput exome and candidate-gene sequencing identified causative mutations in Arhgef12 and Prkci, respectively. Epistasis analysis of both strains indicated that the increased dysplastic phenotype required homozygosity of the Crb1rd8 allele. Retinal dysplastic lesions in Tvrm266 mice were smaller and caused less photoreceptor degeneration than those in Tvrm323 mice, which developed an early, large diffuse lesion phenotype. At one month of age, Müller glia and microglia mislocalization at dysplastic lesions in both modifier strains was similar to that in B6.Cg-Crb1rd8/Pjn mice but photoreceptor cell mislocalization was more extensive. External limiting membrane disruption was comparable in Tvrm266 and B6.Cg-Crb1rd8/Pjn mice but milder in Tvrm323 mice. Immunohistological analysis of mice at postnatal day 0 indicated a normal distribution of mitotic cells in Tvrm266 and Tvrm323 mice, suggesting normal early development. Aberrant electroretinography responses were observed in both models but functional decline was significant only in Tvrm323 mice. These results identify Arhgef12 and Prkci as modifier genes that differentially shape Crb1-associated retinal disease, which may be relevant to understanding clinical variability and underlying disease mechanisms in humans.


Assuntos
Proteínas do Tecido Nervoso , Displasia Retiniana , Fatores de Troca de Nucleotídeo Guanina Rho , Animais , Modelos Animais de Doenças , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Retina/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Displasia Retiniana/genética , Displasia Retiniana/metabolismo , Displasia Retiniana/patologia , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo
3.
PLoS One ; 17(3): e0254469, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35239671

RESUMO

Chediak-Higashi syndrome, caused by mutations in the Lysosome Trafficking Regulator (Lyst) gene, is a recessive hypopigmentation disorder characterized by albinism, neuropathies, neurodegeneration, and defective immune responses, with enlargement of lysosomes and lysosome-related organelles. Although recent studies have suggested that Lyst mutations impair the regulation of sizes of lysosome and lysosome-related organelle, the underlying pathogenic mechanism of Chediak-Higashi syndrome is still unclear. Here we show striking evidence that deficiency in LYST protein function leads to accumulation of photoreceptor outer segment phagosomes in retinal pigment epithelial cells, and reduces adhesion between photoreceptor outer segment and retinal pigment epithelial cells in a mouse model of Chediak-Higashi syndrome. In addition, we observe elevated levels of cathepsins, matrix metallopeptidase (MMP) 3 and oxidative stress markers in the retinal pigment epithelium of Lyst mutants. Previous reports showed that impaired degradation of photoreceptor outer segment phagosomes causes elevated oxidative stress, which could consequently lead to increases of cysteine cathepsins and MMPs in the extracellular matrix. Taken together, we conclude that the loss of LYST function causes accumulation of phagosomes in the retinal pigment epithelium and elevation of several extracellular matrix-remodeling proteases through oxidative stress, which may, in turn, reduce retinal adhesion. Our work reveals previously unreported pathogenic events in the retinal pigment epithelium caused by Lyst deficiency. The same pathogenic events may be conserved in other professional phagocytic cells, such as macrophages in the immune system, contributing to overall Chediak-Higashi syndrome pathology.


Assuntos
Peptídeo Hidrolases
4.
Int J Mol Sci ; 23(4)2022 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-35216333

RESUMO

Fluid and solute transporters of the retinal pigment epithelium (RPE) are core components of the outer blood-retinal barrier. Characterizing these transporters and their role in retinal homeostasis may provide insights into ocular function and disease. Here, we describe RPE defects in tvrm77 mice, which exhibit hypopigmented patches in the central retina. Mapping and nucleotide sequencing of tvrm77 mice revealed a disrupted 5' splice donor sequence in Slc4a5, a sodium bicarbonate cotransporter gene. Slc4a5 expression was reduced 19.7-fold in tvrm77 RPE relative to controls, and alternative splice variants were detected. SLC4A5 was localized to the Golgi apparatus of cultured human RPE cells and in apical and basal membranes. Fundus imaging, optical coherence tomography, microscopy, and electroretinography (ERG) of tvrm77 mice revealed retinal detachment, hypopigmented patches corresponding to neovascular lesions, and retinal folds. Detachment worsened and outer nuclear layer thickness decreased with age. ERG a- and b-wave response amplitudes were initially normal but declined in older mice. The direct current ERG fast oscillation and light peak were reduced in amplitude at all ages, whereas other RPE-associated responses were unaffected. These results link a new Slc4a5 mutation to subretinal fluid accumulation and altered light-evoked RPE electrophysiological responses, suggesting that SLC4A5 functions at the outer blood-retinal barrier.


Assuntos
Mutação/genética , Splicing de RNA/genética , Retina/patologia , Descolamento Retiniano/genética , Epitélio Pigmentado da Retina/patologia , Simportadores de Sódio-Bicarbonato/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Descolamento Retiniano/patologia , Tomografia de Coerência Óptica/métodos
5.
Cell Death Dis ; 12(11): 1017, 2021 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-34716303

RESUMO

Glaucoma is a leading cause of blindness, affecting 70 million people worldwide. Owing to the similarity in anatomy and physiology between human and mouse eyes and the ability to genetically manipulate mice, mouse models are an invaluable resource for studying mechanisms underlying disease phenotypes and for developing therapeutic strategies. Here, we report the discovery of a new mouse model of early-onset glaucoma that bears a transversion substitution c. G344T, which results in a missense mutation, p. R115L in PITX2. The mutation causes an elevation in intraocular pressure (IOP) and progressive death of retinal ganglion cells (RGC). These ocular phenotypes recapitulate features of pathologies observed in human glaucoma. Increased oxidative stress was evident in the inner retina. We demonstrate that the mutant PITX2 protein was not capable of binding to Nuclear factor-like 2 (NRF2), which regulates Pitx2 expression and nuclear localization, and to YAP1, which is necessary for co-initiation of transcription of downstream targets. PITX2-mediated transcription of several antioxidant genes were also impaired. Treatment with N-Acetyl-L-cysteine exerted a profound neuroprotective effect on glaucoma-associated neuropathies, presumably through inhibition of oxidative stress. Our study demonstrates that a disruption of PITX2 leads to glaucoma optic pathogenesis and provides a novel early-onset glaucoma model that will enable elucidation of mechanisms underlying the disease as well as to serve as a resource to test new therapeutic strategies.


Assuntos
Glaucoma/genética , Glaucoma/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mutação de Sentido Incorreto , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAP/metabolismo , Animais , Apoptose/genética , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Pressão Intraocular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Células Ganglionares da Retina/metabolismo , Transfecção , Proteína Homeobox PITX2
6.
Hum Mol Genet ; 27(19): 3340-3352, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29947801

RESUMO

Photoreceptor dysplasia, characterized by formation of folds and (pseudo-)rosettes in the outer retina, is associated with loss of functional nuclear receptor subfamily 2 group E member 3 (NR2E3) and neural retina leucine-zipper (NRL) in both humans and mice. A sensitized chemical mutagenesis study to identify genetic modifiers that suppress photoreceptor dysplasia in Nr2e3rd7mutant mice identified line Tvrm222, which exhibits a normal fundus appearance in the presence of the rd7 mutation. The Tvrm222 modifier of Nr2e3rd7/rd7 was localized to Chromosome 6 and identified as a missense mutation in the FERM domain containing 4B (Frmd4b) gene. The variant is predicted to cause the substitution of a serine residue 938 with proline (S938P). The Frmd4bTvrm222 allele was also found to suppress outer nuclear layer (ONL) rosettes in Nrl-/- mice. Fragmentation of the external limiting membrane (ELM), normally observed in rd7 and Nrl-/-mouse retinas, was absent in the presence of the Frmd4bTvrm222 allele. FRMD4B, a binding partner of cytohesin 3, is proposed to participate in cell junction remodeling. Its biological function in photoreceptor dysplasia has not been previously examined. In vitro experiments showed that the FRMD4B938P variant fails to be efficiently recruited to the cell surface upon insulin stimulation. In addition, we found a reduction in protein kinase B phosphorylation and increased levels of cell junction proteins, Catenin beta 1 and tight junction protein 1, associated with the cell membrane in Tvrm222 retinas. Taken together, this study reveals a critical role of FRMD4B in maintaining ELM integrity and in rescuing morphological abnormalities of the ONL in photoreceptor dysplasia.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Oftalmopatias Hereditárias/genética , Proteínas do Olho/genética , Receptores Nucleares Órfãos/genética , Degeneração Retiniana/genética , Transtornos da Visão/genética , Animais , Oftalmopatias Hereditárias/metabolismo , Oftalmopatias Hereditárias/patologia , Fundo de Olho , Humanos , Camundongos , Mutação de Sentido Incorreto , Domínios Proteicos/genética , Retina/crescimento & desenvolvimento , Retina/patologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Segmento Externo das Células Fotorreceptoras da Retina , Transtornos da Visão/metabolismo , Transtornos da Visão/patologia
7.
Mol Ther ; 26(6): 1581-1593, 2018 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-29673930

RESUMO

Most genetically distinct inherited retinal degenerations are primary photoreceptor degenerations. We selected a severe early onset form of Leber congenital amaurosis (LCA), caused by mutations in the gene LCA5, in order to test the efficacy of gene augmentation therapy for a ciliopathy. The LCA5-encoded protein, Lebercilin, is essential for the trafficking of proteins and vesicles to the photoreceptor outer segment. Using the AAV serotype AAV7m8 to deliver a human LCA5 cDNA into an Lca5 null mouse model of LCA5, we show partial rescue of retinal structure and visual function. Specifically, we observed restoration of rod-and-cone-driven electroretinograms in about 25% of injected eyes, restoration of pupillary light responses in the majority of treated eyes, an ∼20-fold decrease in target luminance necessary for visually guided behavior, and improved retinal architecture following gene transfer. Using LCA5 patient-derived iPSC-RPEs, we show that delivery of the LCA5 cDNA restores lebercilin protein and rescues cilia quantity. The results presented in this study support a path forward aiming to develop safety and efficacy trials for gene augmentation therapy in human subjects with LCA5 mutations. They also provide the framework for measuring the effects of intervention in ciliopathies and other severe, early-onset blinding conditions.


Assuntos
Cegueira/metabolismo , Cegueira/terapia , Dependovirus/genética , Terapia Genética/métodos , Animais , Eletrorretinografia , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Humanos , Amaurose Congênita de Leber/metabolismo , Amaurose Congênita de Leber/terapia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo
8.
Mol Vis ; 23: 140-148, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28356706

RESUMO

PURPOSE: Familial exudative vitreoretinopathy (FEVR) is caused by mutations in the genes encoding low-density lipoprotein receptor-related protein (LRP5) or its interacting partners, namely frizzled class receptor 4 (FZD4) and norrin cystine knot growth factor (NDP). Mouse models for Lrp5, Fzd4, and Ndp have proven to be important for understanding the retinal pathophysiology underlying FEVR and systemic abnormalities related to defective Wnt signaling. Here, we report a new mouse mutant, tvrm111B, which was identified by electroretinogram (ERG) screening of mice generated in the Jackson Laboratory Translational Vision Research Models (TVRM) mutagenesis program. METHODS: ERGs were used to examine outer retinal physiology. The retinal vasculature was examined by in vivo retinal imaging, as well as by histology and immunohistochemistry. The tvrm111B locus was identified by genetic mapping of mice generated in a cross to DBA/2J, and subsequent sequencing analysis. Gene expression was examined by real-time PCR of retinal RNA. Bone mineral density (BMD) was examined by peripheral dual-energy X-ray absorptiometry. RESULTS: The tvrm111B allele is inherited as an autosomal recessive trait. Genetic mapping of the decreased ERG b-wave phenotype of tvrm111B mice localized the mutation to a region on chromosome 19 that included Lrp5. Sequencing of Lrp5 identified the insertion of a cytosine (c.4724_4725insC), which is predicted to cause a frameshift that disrupts the last three of five conserved PPPSPxS motifs in the cytoplasmic domain of LRP5, culminating in a premature termination. In addition to a reduced ERG b-wave, Lrp5tvrm111B homozygotes have low BMD and abnormal features of the retinal vasculature that have been reported previously in Lrp5 mutant mice, including persistent hyaloid vessels, leakage on fluorescein angiography, and an absence of the deep retinal capillary bed. CONCLUSIONS: The phenotype of the Lrp5tvrm111B mutant includes abnormalities of the retinal vasculature and of BMD. This model may be a useful resource to further our understanding of the biological role of LRP5 and to evaluate experimental therapies for FEVR or other conditions associated with LRP5 dysfunction.


Assuntos
Densidade Óssea , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Mutagênese/genética , Mutação/genética , Vasos Retinianos/anormalidades , Vasos Retinianos/fisiopatologia , Animais , Eletrorretinografia , Regulação da Expressão Gênica , Homozigoto , Masculino , Camundongos Endogâmicos C57BL , Tamanho do Órgão/genética , Fenótipo , Vasos Retinianos/diagnóstico por imagem , Vasos Retinianos/patologia , Via de Sinalização Wnt/genética
9.
Am J Pathol ; 186(7): 1925-1938, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27207593

RESUMO

The nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) enzyme is essential for regenerating the nuclear pool of NAD(+) in all nucleated cells in the body, and mounting evidence also suggests that it has a separate role in neuroprotection. Recently, mutations in the NMNAT1 gene were associated with Leber congenital amaurosis, a severe retinal degenerative disease that causes blindness during infancy. Availability of a reliable mammalian model of NMNAT1-Leber congenital amaurosis would assist in determining the mechanisms through which disruptions in NMNAT1 lead to retinal cell degeneration and would provide a resource for testing treatment options. To this end, we identified two separate N-ethyl-N-nitrosourea-generated mouse lines that harbor either a p.V9M or a p.D243G mutation. Both mouse models recapitulate key aspects of the human disease and confirm the pathogenicity of mutant NMNAT1. Homozygous Nmnat1 mutant mice develop a rapidly progressing chorioretinal disease that begins with photoreceptor degeneration and includes attenuation of the retinal vasculature, optic atrophy, and retinal pigment epithelium loss. Retinal function deteriorates in both mouse lines, and, in the more rapidly progressing homozygous Nmnat1(V9M) mutant mice, the electroretinogram becomes undetectable and the pupillary light response weakens. These mouse models offer an opportunity for investigating the cellular mechanisms underlying disease pathogenesis, evaluating potential therapies for NMNAT1-Leber congenital amaurosis, and conducting in situ studies on NMNAT1 function and NAD(+) metabolism.


Assuntos
Modelos Animais de Doenças , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/fisiopatologia , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Animais , Genótipo , Humanos , Camundongos , Camundongos Mutantes , Reação em Cadeia da Polimerase
10.
Invest Ophthalmol Vis Sci ; 55(1): 387-95, 2014 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-24346171

RESUMO

PURPOSE: We directly corrected the mouse Crb1(rd8) gene mutation, which is present in many inbred laboratory strains derived from C57BL/6N and complicates genetic studies of retinal disease in mice. METHODS: Fertilized C57BL/6NJ oocytes were coinjected with mRNAs encoding a transcription activator-like effector nuclease (TALEN) targeting the Crb1(rd8) allele plus single-stranded oligonucleotides to correct the allele. The oligonucleotides included additional nucleotide changes to distinguish the corrected allele (Crb1(em1Mvw)) from wild-type Crb1 and to minimize TALEN recutting. Oligonucleotide length, concentration of injected oligonucleotides and TALEN mRNAs were varied to optimize homology-directed repair of the locus. Following microinjection, embryos were carried to term in pseudopregnant females. Correction efficiency was assessed by PCR analysis of the Crb1(em1Mvw) allele. Phenotypic correction was demonstrated by fundus imaging and optical coherence tomography of live mice, and by confocal fluorescence microscopy of retinal flat mounts. RESULTS: Under optimal conditions, homology-directed repair was observed in 27% (8/30) of live-born animals and showed minimal illegitimate recombination of donor DNA. However, extensive founder mosaicism was evident, emphasizing the need to analyze offspring of founder animals. Unlike C57BL/6NJ mice, which exhibited external limiting membrane fragmentation and regional retinal dysplasia, heterozygous Crb1(em1Mvw)/Crb1(rd8) mice showed a normal retinal phenotype. CONCLUSIONS: The C57BL/6NJ-Crb1(rd8) mutation and its associated retinal phenotypes were corrected efficiently by TALEN-mediated homology-directed repair. The C57BL/6NJ-Crb1(em1Mvw) mice generated by this strategy will enhance ocular phenotyping efforts based on the C57BL/6N background, such as those implemented by the International Mouse Phenotyping Consortium (IMPC) project.


Assuntos
Camundongos Endogâmicos C57BL/genética , Mutação , Proteínas do Tecido Nervoso/genética , Reparo de DNA por Recombinação/genética , Retina/metabolismo , Degeneração Retiniana/genética , Alelos , Animais , Modelos Animais de Doenças , Células-Tronco Embrionárias/metabolismo , Proteínas do Olho , Feminino , Terapia Genética/métodos , Camundongos , Microscopia Confocal , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Reação em Cadeia da Polimerase , Retina/patologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/terapia , Tomografia de Coerência Óptica
11.
Hum Mol Genet ; 22(3): 558-67, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23108158

RESUMO

Approximately 36 000 cases of simplex and familial retinitis pigmentosa (RP) worldwide are caused by a loss in phosphodiesterase (PDE6) function. In the preclinical Pde6α(nmf363) mouse model of this disease, defects in the α-subunit of PDE6 result in a progressive loss of photoreceptors and neuronal function. We hypothesized that increasing PDE6α levels using an AAV2/8 gene therapy vector could improve photoreceptor survival and retinal function. We utilized a vector with the cell-type-specific rhodopsin (RHO) promoter: AAV2/8(Y733F)-Rho-Pde6α, to transduce Pde6α(nmf363) retinas and monitored its effects over a 6-month period (a quarter of the mouse lifespan). We found that a single injection enhanced survival of photoreceptors and improved retinal function. At 6 months of age, the treated eyes retained photoreceptor cell bodies, while there were no detectable photoreceptors remaining in the untreated eyes. More importantly, the treated eyes demonstrated functional visual responses even after the untreated eyes had lost all vision. Despite focal rescue of the retinal structure adjacent to the injection site, global functional rescue of the entire retina was observed. These results suggest that RP due to PDE6α deficiency in humans, in addition to PDE6ß deficiency, is also likely to be treatable by gene therapy.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Terapia Genética/métodos , Retinose Pigmentar/genética , Retinose Pigmentar/terapia , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Eletrorretinografia , Vetores Genéticos , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regiões Promotoras Genéticas , Retina/anatomia & histologia , Retina/fisiopatologia , Retinose Pigmentar/fisiopatologia , Rodopsina/genética , Rodopsina/metabolismo , Transdução Genética
12.
J Clin Invest ; 121(6): 2169-80, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21606596

RESUMO

The mutations that cause Leber congenital amaurosis (LCA) lead to photoreceptor cell death at an early age, causing childhood blindness. To unravel the molecular basis of LCA, we analyzed how mutations in LCA5 affect the connectivity of the encoded protein lebercilin at the interactome level. In photoreceptors, lebercilin is uniquely localized at the cilium that bridges the inner and outer segments. Using a generally applicable affinity proteomics approach, we showed that lebercilin specifically interacted with the intraflagellar transport (IFT) machinery in HEK293T cells. This interaction disappeared when 2 human LCA-associated lebercilin mutations were introduced, implicating a specific disruption of IFT-dependent protein transport, an evolutionarily conserved basic mechanism found in all cilia. Lca5 inactivation in mice led to partial displacement of opsins and light-induced translocation of arrestin from photoreceptor outer segments. This was consistent with a defect in IFT at the connecting cilium, leading to failure of proper outer segment formation and subsequent photoreceptor degeneration. These data suggest that lebercilin functions as an integral element of selective protein transport through photoreceptor cilia and provide a molecular demonstration that disrupted IFT can lead to LCA.


Assuntos
Proteínas do Olho/fisiologia , Amaurose Congênita de Leber/fisiopatologia , Proteínas Associadas aos Microtúbulos/fisiologia , Cílio Conector dos Fotorreceptores/fisiologia , Transporte Proteico/fisiologia , Animais , Arrestinas/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Proteínas do Olho/genética , Humanos , Amaurose Congênita de Leber/genética , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Complexos Multiproteicos , Opsinas/metabolismo , Mapeamento de Interação de Proteínas , Transporte Proteico/genética , Proteínas Recombinantes de Fusão/fisiologia , Segmento Externo da Célula Bastonete/metabolismo , Segmento Externo da Célula Bastonete/patologia , Visão Ocular/fisiologia
13.
Invest Ophthalmol Vis Sci ; 52(7): 4703-9, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21436282

RESUMO

PURPOSE: To determine the basis and to characterize the phenotype of a chemically induced mutation in a mouse model of retinal degeneration. METHODS: Screening by indirect ophthalmoscopy identified a line of N-ethyl-N-nitrosourea (ENU) mutagenized mice demonstrating retinal patches. Longitudinal studies of retinal histologic sections showed photoreceptors in the peripheral retina undergoing slow, progressive degeneration. The mutation was named neuroscience mutagenesis facility 12 (nmf12), and mapping localized the critical region to Chromosome 2. RESULTS: Sequencing of nmf12 DNA revealed a point mutation in the c-mer tyrosine kinase gene, designated Mertk(nmf12). We detected elevated levels of tumor necrosis factor (Tnf, previously Tnfa) in retinas of Mertk(nmf12) homozygotes relative to wild-type controls and investigated whether the increase of TNF, an inflammatory cytokine produced by macrophages/monocytes that signals intracellularly to cause necrosis or apoptosis, could underlie the retinal degeneration observed in Mertk(nmf12) homozygotes. Mertk(nmf12) homozygous mice were mated to mice lacking the entire Tnf gene and partial coding sequences of the Lta (Tnfb) and Ltb (Tnfc) genes.(2) B6.129P2-Ltb/Tnf/Lta(tm1Dvk)/J homozygotes did not exhibit a retinal degeneration phenotype and will, hereafter, be referred to as Tnfabc(-/-) mice. Surprisingly, mice homozygous for both the Mertk(nmf12) and the Ltb/Tnf/Lta(tm1Dvk) allele (Tnfabc(-/-)) demonstrated an increase in the rate of retinal degeneration. CONCLUSIONS: These findings illustrate that a mutation in the Mertk gene leads to a significantly slower progressive retinal degeneration compared with other alleles of Mertk. These results demonstrate that TNF family members play a role in protecting photoreceptors of Mertk(nmf12) homozygotes from cell death.


Assuntos
DNA/genética , Etilnitrosoureia/toxicidade , Mutação/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Retina/metabolismo , Degeneração Retiniana/genética , Animais , Western Blotting , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Modelos Animais de Doenças , Progressão da Doença , Eletrorretinografia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Oftalmoscopia , Fenótipo , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Proteínas Proto-Oncogênicas/biossíntese , Receptores Proteína Tirosina Quinases/biossíntese , Retina/efeitos dos fármacos , Retina/patologia , Degeneração Retiniana/patologia , Degeneração Retiniana/fisiopatologia , Fator de Necrose Tumoral alfa/biossíntese , c-Mer Tirosina Quinase
14.
J Ophthalmol ; 2011: 391384, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21052544

RESUMO

The need for mouse models, with their well-developed genetics and similarity to human physiology and anatomy, is clear and their central role in furthering our understanding of human disease is readily apparent in the literature. Mice carrying mutations that alter developmental pathways or cellular function provide model systems for analyzing defects in comparable human disorders and for testing therapeutic strategies. Mutant mice also provide reproducible, experimental systems for elucidating pathways of normal development and function. Two programs, the Eye Mutant Resource and the Translational Vision Research Models, focused on providing such models to the vision research community are described herein. Over 100 mutant lines from the Eye Mutant Resource and 60 mutant lines from the Translational Vision Research Models have been developed. The ocular diseases of the mutant lines include a wide range of phenotypes, including cataracts, retinal dysplasia and degeneration, and abnormal blood vessel formation. The mutations in disease genes have been mapped and in some cases identified by direct sequencing. Here, we report 3 novel alleles of Crx(tvrm65), Rp1(tvrm64), and Rpe65(tvrm148) as successful examples of the TVRM program, that closely resemble previously reported knockout models.

15.
Hum Mol Genet ; 20(3): 482-96, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21078623

RESUMO

Nephronophthisis (NPHP) is an autosomal recessive kidney disease that is often associated with vision and/or brain defects. To date, 11 genes are known to cause NPHP. The gene products, while structurally unrelated, all localize to cilia or centrosomes. Although mouse models of NPHP are available for 9 of the 11 genes, none has been described for nephronophthisis 4 (Nphp4). Here we report a novel, chemically induced mutant, nmf192, that bears a nonsense mutation in exon 4 of Nphp4. Homozygous mutant Nphp4(nmf192/nmf192) mice do not exhibit renal defects, phenotypes observed in human patients bearing mutations in NPHP4, but they do develop severe photoreceptor degeneration and extinguished rod and cone ERG responses by 9 weeks of age. Photoreceptor outer segments (OS) fail to develop properly, and some OS markers mislocalize to the inner segments and outer nuclear layer in the Nphp4(nmf192/nmf192) mutant retina. Despite NPHP4 localization to the transition zone in the connecting cilia (CC), the CC appear to be normal in structure and ciliary transport function is partially retained. Likewise, synaptic ribbons develop normally but then rapidly degenerate by P14. Finally, Nphp4(nmf192/nmf192) male mutants are sterile and show reduced sperm motility and epididymal sperm counts. Although Nphp4(nmf192/nmf192) mice fail to recapitulate the kidney phenotype of NPHP, they will provide a valuable tool to further elucidate how NPHP4 functions in the retina and male reproductive organs.


Assuntos
Células Fotorreceptoras de Vertebrados/fisiologia , Células Fotorreceptoras de Vertebrados/ultraestrutura , Proteínas/genética , Proteínas/fisiologia , Degeneração Retiniana/genética , Maturação do Esperma/genética , Sinapses/fisiologia , Sinapses/ultraestrutura , Animais , Western Blotting , Mapeamento Cromossômico , Cílios/metabolismo , Códon sem Sentido , Eletrorretinografia , Infertilidade Masculina/genética , Doenças Renais Císticas/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Compostos de Nitrosoureia/farmacologia , Fenótipo , Retina/anormalidades , Análise de Sequência de DNA , Motilidade dos Espermatozoides
16.
J Biol Chem ; 285(10): 7697-711, 2010 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-20048158

RESUMO

The Neuromutagenesis Facility at the Jackson Laboratory generated a mouse model of retinal vasculopathy, nmf223, which is characterized clinically by vitreal fibroplasia and vessel tortuosity. nmf223 homozygotes also have reduced electroretinogram responses, which are coupled histologically with a thinning of the inner nuclear layer. The nmf223 locus was mapped to chromosome 17, and a missense mutation was identified in Lama1 that leads to the substitution of cysteine for a tyrosine at amino acid 265 of laminin alpha1, a basement membrane protein. Despite normal localization of laminin alpha1 and other components of the inner limiting membrane, a reduced integrity of this structure was suggested by ectopic cells and blood vessels within the vitreous. Immunohistochemical characterization of nmf223 homozygous retinas demonstrated the abnormal migration of retinal astrocytes into the vitreous along with the persistence of hyaloid vasculature. The Y265C mutation significantly reduced laminin N-terminal domain (LN) interactions in a bacterial two-hybrid system. Therefore, this mutation could affect interactions between laminin alpha1 and other laminin chains. To expand upon these findings, a Lama1 null mutant, Lama1(tm1.1Olf), was generated that exhibits a similar but more severe retinal phenotype than that seen in nmf223 homozygotes. The increased severity of the Lama1 null mutant phenotype is probably due to the complete loss of the inner limiting membrane in these mice. This first report of viable Lama1 mouse mutants emphasizes the importance of this gene in retinal development. The data presented herein suggest that hypomorphic mutations in human LAMA1 could lead to retinal disease.


Assuntos
Laminina , Mutação de Sentido Incorreto , Isoformas de Proteínas , Retina , Doenças Retinianas , Vasos Retinianos , Adulto , Sequência de Aminoácidos , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Membrana Basal/citologia , Membrana Basal/metabolismo , Eletrorretinografia , Feminino , Teste de Complementação Genética , Humanos , Laminina/genética , Laminina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Fenótipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Retina/anormalidades , Retina/anatomia & histologia , Retina/fisiologia , Doenças Retinianas/genética , Doenças Retinianas/patologia , Vasos Retinianos/anormalidades , Vasos Retinianos/anatomia & histologia , Vasos Retinianos/fisiologia , Alinhamento de Sequência , Transgenes
17.
Mol Endocrinol ; 22(8): 1866-80, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18483174

RESUMO

Smallie (slie), a spontaneous, autosomal-recessive mutation causes dwarfing and infertility in mice. The purpose of this study was to determine and characterize the underlying molecular genetic basis for its phenotype. The slie locus was mapped to chromosome 1, and fine-structure mapping narrowed the slie allele within 2 Mb between genetic markers D1Mit36 and Mpz. To pinpoint the underlying mutation quantitative real-time PCR was used to measure the relative expression levels for the genes residing within this region. Expression of one gene, Ddr2, which encodes discoidin domain receptor 2 (DDR2), was absent in slie homozygote mice. Genomic sequencing analysis detected a 150-kb deletion that extended into the Ddr2 gene transcript. Detailed phenotype analysis revealed that gonadal dysregulation underlies infertility in slie mice because all females were anovulatory and most adult males lacked spermatogenesis. The pituitary gland of prepubertal slie mice was smaller than in wild-type mice. The basal levels and gene expression for pituitary and hypothalamic hormones, and gene expression for hypothalamic-releasing hormones, were not significantly different between slie and wild-type mice. Circulating levels of IGF-1 did not differ in slie mice despite lower Igf-1 mRNA expression in the liver. After exogenous gonadotropin administration, the levels of secreted steroid hormones in both male and female adult slie mice were blunted compared to adult wild-type, but was similar to prepubertal wild-type mice. Taken together, our results indicate that the absence of DDR2 leads to growth retardation and gonadal dysfunction due to peripheral defects in hormonal-responsive pathways in slie mice.


Assuntos
Alelos , Nanismo/complicações , Nanismo/genética , Transtornos Gonadais/complicações , Transtornos Gonadais/genética , Receptores Proteína Tirosina Quinases/genética , Receptores de Colágeno/genética , Receptores Mitogênicos/genética , Animais , Composição Corporal , Mapeamento Cromossômico , Clonagem Molecular , Receptores com Domínio Discoidina , Feminino , Fertilidade , Regulação da Expressão Gênica no Desenvolvimento , Gônadas/anormalidades , Gônadas/patologia , Fator de Crescimento Insulin-Like I/metabolismo , Longevidade , Masculino , Camundongos , Camundongos Mutantes , Mutação/genética , Sistemas Neurossecretores/metabolismo , Hormônios Peptídicos/sangue , Hormônios Peptídicos/genética , Hormônios Peptídicos/metabolismo , Fenótipo , Hipófise/patologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Colágeno/metabolismo , Receptores Mitogênicos/metabolismo
18.
Mamm Genome ; 19(3): 145-54, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18286335

RESUMO

The retinal degeneration 7 (rd7) mouse, lacking expression of the Nr2e3 gene, exhibits retinal dysplasia and a slow, progressive degeneration due to an abnormal production of blue opsin-expressing cone cells. In this study we evaluated three strains of mice to identify alleles that would slow or ameliorate the retinal degeneration observed in Nr2e3 (rd7/rd7) mice. Our studies reveal that genetic background greatly influences the expression of the Nr2e3 (rd7/rd7) phenotype and that the inbred mouse strains CAST/EiJ, AKR/J, and NOD.NON-H2 (nb1) carry alleles that confer resistance to Nr2e3 (rd7/rd7)-induced retinal degeneration. B6.Cg-Nr2e3 (rd7/rd7) mice were outcrossed to each strain and the F(1) progeny were intercrossed to produce F(2) mice. In each intercross, 20-24% of the total F(2) progeny were homozygous for the Nr2e3 (rd7/rd7) mutation in a mixed genetic background; approximately 28-48% of the Nr2e3 (rd7/rd7) homozygotes were suppressed for the degenerative retina phenotype in a mixed genetic background. The suppressed mice had no retinal spots and normal retinal morphology with a normal complement of blue opsin-expressing cone cells. An initial genome scan revealed a significant association of the suppressed phenotype with loci on chromosomes 8 and 19 with the CAST/EiJ background, two marginal loci on chromosomes 7 and 11 with the AKR/J background, and no significant QTL with the NOD.NON-H2 (nb1) background. We did not observe any significant epistatic effects in this study. Our results suggest that there are several genes that are likely to act in the same or parallel pathway as NR2E3 that can rescue the Nr2e3 (rd7/rd7) phenotype and may serve as potential therapeutic targets.


Assuntos
Receptores Citoplasmáticos e Nucleares/genética , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/genética , Animais , Cruzamentos Genéticos , Epistasia Genética , Camundongos , Mutação , Receptores Nucleares Órfãos , Receptores Citoplasmáticos e Nucleares/metabolismo , Degeneração Retiniana/patologia , Especificidade da Espécie
19.
Mol Cell Neurosci ; 35(1): 161-70, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17376701

RESUMO

Carbonic anhydrase related protein 8 (Car8) is known to be abundantly expressed in Purkinje cells (PCs), and its genetic mutation causes a motor coordination defect. To determine the underlying mechanism, we analyzed the mouse cerebellum carrying a Car8 mutation. Electrophysiological analysis showed that spontaneous excitatory transmission was largely diminished while paired pulse ratio at parallel fiber-PC synapses was comparable to wild-type, suggesting functional synapses have normal release probability but the number of functional synapses may be lower in mutants. Light microscopic study revealed an abnormal extension of climbing fibers to the distal PC dendrites. At the ultrastructural level, we found numerous PC spines not forming synapses primarily in distal dendrites and occasionally multiple spines contacting a single varicosity. These abnormalities of parallel fiber-PC synapses may underlie the functional defect in excitatory transmission. Thus, Car8 plays a critical role in synaptogenesis and/or maintenance of proper synaptic morphology and function in the cerebellum.


Assuntos
Biomarcadores Tumorais/genética , Doenças Cerebelares/patologia , Doenças Cerebelares/fisiopatologia , Proteínas do Tecido Nervoso/genética , Células de Purkinje/patologia , Sinapses/patologia , Animais , Biomarcadores Tumorais/fisiologia , Doenças Cerebelares/genética , Cerebelo/patologia , Cerebelo/fisiologia , Espinhas Dendríticas/enzimologia , Espinhas Dendríticas/patologia , Espinhas Dendríticas/ultraestrutura , Potenciais Pós-Sinápticos Excitadores/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Mutantes Neurológicos , Microscopia Eletrônica , Proteínas do Tecido Nervoso/fisiologia , Células de Purkinje/fisiologia , Células de Purkinje/ultraestrutura , Sinapses/fisiologia , Sinapses/ultraestrutura
20.
Physiol Genomics ; 27(2): 131-40, 2006 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-16849632

RESUMO

Tub is a member of a small gene family, the tubby-like proteins (TULPs), with predominant expression in neurons. Mice carrying a mutation in Tub develop retinal and cochlear degeneration as well as late-onset obesity with insulin resistance. During behavioral and metabolic testing, we found that homozygous C57BL/6J-Tub(tub) mice have a lower respiratory quotient than C57BL/6J controls before the onset of obesity, indicating that tubby homozygotes fail to activate carbohydrate metabolism and instead rely on fat metabolism for energy needs. In concordance with this, tubby mice show higher excretion of ketone bodies and accumulation of glycogen in the liver. Quantitation of liver mRNA levels shows that, during the transition from light to dark period, tubby mice fail to induce glucose-6-phosphate dehydrogenase (G6pdh), the rate-limiting enzyme in the pentose phosphate pathway that normally supplies NADPH for de novo fatty acid synthesis and glutathione reduction. Reduced G6PDH protein levels and enzymatic activity in tubby mice lead accordingly to lower levels of NADPH and reduced glutathione (GSH), respectively. mRNA levels for the lipolytic enzymes acetyl-CoA synthetase and carnitine palmitoyltransferase are increased during the dark cycle and decreased during the light period, and several citric acid cycle genes are dysregulated in tubby mice. Examination of hypothalamic gene expression showed high levels of preproorexin mRNA leading to accumulation of orexin peptide in the lateral hypothalamus. We hypothesize that abnormal hypothalamic orexin expression leads to changes in liver carbohydrate metabolism and may contribute to the moderate obesity observed in tubby mice.


Assuntos
Metabolismo dos Carboidratos/genética , Metabolismo Energético/genética , Camundongos Mutantes/metabolismo , Proteínas/genética , Acetato-CoA Ligase/biossíntese , Acetato-CoA Ligase/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteína Relacionada com Agouti , Animais , Química Encefálica , Dióxido de Carbono/metabolismo , Carnitina O-Palmitoiltransferase/biossíntese , Carnitina O-Palmitoiltransferase/genética , Ritmo Circadiano , Ciclo do Ácido Cítrico/genética , Doenças Cocleares/genética , Ingestão de Alimentos , Indução Enzimática/genética , Genes Recessivos , Glucosefosfato Desidrogenase/biossíntese , Glucosefosfato Desidrogenase/genética , Glutationa/deficiência , Homozigoto , Hipotálamo/metabolismo , Resistência à Insulina/genética , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Metabolismo dos Lipídeos , Lipólise/genética , Fígado/metabolismo , Glicogênio Hepático/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes/genética , Atividade Motora , NADP/deficiência , Neuropeptídeo Y/biossíntese , Neuropeptídeo Y/genética , Neuropeptídeos/biossíntese , Neuropeptídeos/genética , Obesidade/genética , Orexinas , Oxigênio/metabolismo , Consumo de Oxigênio/genética , Via de Pentose Fosfato/genética , Proteínas/fisiologia , Degeneração Retiniana/genética
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