RESUMO
Kinesin and dynein are opposite-polarity microtubule motors that drive the tightly regulated transport of a variety of cargoes. Both motors can bind to cargo, but their overall composition on axonal vesicles and whether this composition directly modulates transport activity are unknown. Here we characterize the intracellular transport and steady-state motor subunit composition of mammalian prion protein (PrP(C)) vesicles. We identify Kinesin-1 and cytoplasmic dynein as major PrP(C) vesicle motor complexes and show that their activities are tightly coupled. Regulation of normal retrograde transport by Kinesin-1 is independent of dynein-vesicle attachment and requires the vesicle association of a complete Kinesin-1 heavy and light chain holoenzyme. Furthermore, motor subunits remain stably associated with stationary as well as with moving vesicles. Our data suggest a coordination model wherein PrP(C) vesicles maintain a stable population of associated motors whose activity is modulated by regulatory factors instead of by structural changes to motor-cargo associations.
Assuntos
Axônios/metabolismo , Dineínas/metabolismo , Cinesinas/metabolismo , Proteínas PrPC/metabolismo , Animais , Hipocampo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora , Neurônios/metabolismo , Vesículas Transportadoras/metabolismoRESUMO
We have investigated how connexin 46 (Cx46) regulates lens stiffness by studying different Cx46 knockout (Cx46KO) mice. A modified muscle lever system was used to determine the lens stiffness of wild-type (WT) and Cx46KO mice at the C57BL/6J (B6) and the 129SvJae (129) strain backgrounds according to total lens displacement at the point of maximum force when fresh lenses were compressed with a maximum of 2 mN of force. In comparison to B6-WT controls, young and old B6-Cx46KO lenses showed 23% and 28% reductions in lens displacement, respectively. Comparing to 129-WT controls, old 129-Cx46KO lenses showed 50% reduction in the lens displacement while young 129-Cx46KO lenses displayed similar displacement. Old B6-Cx46KO and old 129-Cx46KO lenses showed almost identical lens displacement, 128 µm versus 127 µm. Morphological data revealed unique changes of peripheral fiber cell shapes in young B6-WT lenses but not in young B6-Cx46KO, 129-WT and 129-Cx46KO lenses. This work reveals Cx46 deletion increases the lens stiffness in both young and old mice at B6 strain background but only in old mice at 129 strain background which contains intermediate filament CP49 gene deletion. Cx46 impairment increases old mouse lens stiffness and may contribute to the development of presbyopia.
Assuntos
Envelhecimento/fisiologia , Catarata/fisiopatologia , Conexinas/genética , Cristalino/fisiopatologia , Animais , Catarata/metabolismo , Conexinas/metabolismo , Modelos Animais de Doenças , Elasticidade , Junções Comunicantes , Cristalino/metabolismo , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Purpose: To investigate the molecular and cellular mechanisms of cataract induced by cold temperatures in young lenses of wild-type C57BL/6J (B6), wild-type 129SvJae (129), and filensin knockout (KO) mice. To determine how lens intermediate filament proteins, filensin (BFSP1) and CP49 (BFSP2), are involved in the formation of cold cataract. Methods: The formation of cold cataract was examined in enucleated lenses at different temperatures and was imaged under a dissecting microscope. Lens vibratome sections were prepared, immunostained with different antibodies and fluorescent probes, and then imaged with a laser confocal microscope to evaluate the protein distribution and the membrane and cytoskeleton structures in the lens fibers. Results: Postnatal day 14 (P14) wild-type B6 lenses showed cataracts dependent on cold temperatures in interior fibers about 420-875 µm (zone III) and 245-875 µm (zone II and zone III) from the lens surface, under 25 °C and 4 °C, respectively. In contrast, wild-type 129 (with CP49 gene deletion) and filensin KO (on the B6 background) lenses did not have cold cataracts at 25 °C but displayed a reduced cold cataract, especially in zone III, at 4 °C. Immunofluorescent staining data revealed that CP49 and filensin proteins were uniformly distributed in fiber cell cytosols without cold cataracts but accumulated or aggregated in the cell boundaries of the fibers where cold cataracts appeared. Conclusions: CP49 and filensin are important components for the formation of cold cataract in young B6 mouse lenses. Accumulated or aggregated CP49 and filensin beaded intermediate filaments in fiber cell boundaries might directly or indirectly contribute to the light scattering of cold cataract. Cold cataract in zone II is independent of beaded intermediate filaments. CP49 and filensin intermediate filaments and other lens proteins probably form distinct high molecular organizations to regulate lens transparency in interior fibers.
Assuntos
Catarata/genética , Proteínas do Olho/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Cristalino/metabolismo , Animais , Catarata/metabolismo , Temperatura Baixa , Citoesqueleto/metabolismo , Proteínas do Olho/genética , Feminino , Imuno-Histoquímica , Proteínas de Filamentos Intermediários/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
We studied the role of sodium/proton exchanger 8 (NHE8) in retinal pigment epithelium (RPE) and photoreceptor cells of adult mouse retina by using the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Neisseria meningitidis (Nm). Specific single guide RNAs (sgRNAs) were designed to knockdown the Slc9a8 gene, which encodes the NHE8. Nuclease null NmCas9 and sgRNAs were packaged respectively using adeno-associated viral vector (AAV), and delivered into mouse eyes in vivo by subretinal injection on wild-type mice of about four-week-old when mouse retina is fully developed. Eye samples were collected four weeks after injection for phenotype examination. Real-time PCR analysis demonstrated â¼38% reduction of NHE8 transcripts in retinas injected with AAV-knockdown sgRNA and AAV-Cas9. Loss of photoreceptor cells was found in eyes injected with AAV-knockdown sgRNA and AAV-Cas9 under either the human rhodopsin promoter or the minimal chicken ß-actin promoter, while normal morphology was observed in control eyes injected with AAV-Cas9 and AAV-control sgRNA; immunostaining data showed degenerating photoreceptor cells and RPE cells in eyes injected with knockdown sgRNA and Cas9 AAVs. We further determined that mutant M120K-NHE8 displayed altered intracellular pH regulation in human RPE and primary mouse RPE cells using genetically encoded pH sensor pHluorin and that primary cultured NHE8 mutant RPE cells showed different pH titration curves. These results indicate that NHE8 plays essential function in both RPE and photoreceptor cells. NHE8 dysfunction either in photoreceptor or RPE is sufficient to cause retinal degeneration in adult mice at any age.
Assuntos
Proteína 9 Associada à CRISPR/genética , Dependovirus/genética , Técnicas de Inativação de Genes , Células Fotorreceptoras de Vertebrados/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Trocadores de Sódio-Hidrogênio/fisiologia , Animais , Células Cultivadas , Técnica Indireta de Fluorescência para Anticorpo , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos C57BL , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Transdução GenéticaRESUMO
Connexin 50 (Cx50) mediated signaling is essential for controlling the lens growth and size. Cx50 mutations cause microphthalmia, smaller lenses, and cataracts in humans and animals. These ocular defects have never been investigated in live Cx50 mutant mice by using non-invasive imaging techniques. Here, we report a longitudinal study of the ocular defects in Cx50 knockout (Cx50KO) mice from the ages of 3 weeks to 12 months by using spectral-domain optical coherence tomography (SD-OCT). The anterior chamber depth (ACD), lens thickness (LT), vitreous chamber depth (VCD), and axial length (AL) were measured along the visual axis and adjusted with corresponding refractive indices. The SD-OCT image data confirm age-related reductions of LT and AL in live Cx50KO mice compared to age-matched wild-type (WT) controls, and the reduction values are comparable to the in vitro measurements of Cx50KO eyeballs and lenses reported previously. Moreover, reductions of ACD were observed in Cx50KO mice at all ages studied while VCD changes are statistically insignificant in comparison to the WT controls. Therefore, Cx50KO's microphthalmia with small lens is selectively associated with delayed ACD development but not the vitreous formation. This work supports the notion that lens size and/or growth is important for anterior chamber development.
RESUMO
Papillorenal syndrome (PRS, also known as renal-coloboma syndrome) is an autosomal dominant disease characterized by potentially-blinding congenital optic nerve excavation and congenital kidney abnormalities. Many patients with PRS have mutations in the paired box transcription factor gene, PAX2. Although most mutations in PAX2 are predicted to result in complete loss of one allele's function, three missense mutations have been reported, raising the possibility that more subtle alterations in PAX2 function may be disease-causing. To date, the molecular behaviors of these mutations have not been explored. We describe a novel mouse model of PRS due to a missense mutation in a highly-conserved threonine residue in the paired domain of Pax2 (p.T74A) that recapitulates the ocular and kidney findings of patients. This mutation is in the Pax2 paired domain at the same location as two human missense mutations. We show that all three missense mutations disrupt potentially critical hydrogen bonds in atomic models and result in reduced Pax2 transactivation, but do not affect nuclear localization, steady state mRNA levels, or the ability of Pax2 to bind its DNA consensus sequence. Moreover, these mutations show reduced steady-state levels of Pax2 protein in vitro and (for p.T74A) in vivo, likely by reducing protein stability. These results suggest that hypomorphic alleles of PAX2/Pax2 can lead to significant disease in humans and mice.
Assuntos
Anormalidades Múltiplas/genética , Alelos , Mutação de Sentido Incorreto/genética , Fator de Transcrição PAX2/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Cerebelo/patologia , DNA/metabolismo , Embrião de Mamíferos/patologia , Olho/patologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fator de Transcrição PAX2/química , Fator de Transcrição PAX2/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia Estrutural de Proteína , Síndrome , Fatores de TempoRESUMO
The mammalian ocular lens is an avascular multicellular organ that grows continuously throughout life. Traditionally, its cellular organization is investigated using dissected lenses, which eliminates in vivo environmental and structural support. Here, we demonstrated that two-photon fluorescence microscopy (2PFM) can visualize lens cells in vivo. To maintain subcellular resolution at depth, we employed adaptive optics (AO) to correct aberrations due to ocular and lens tissues, which led to substantial signal and resolution improvements. Imaging lens cells up to 980 µm deep, we observed novel cellular organizations including suture-associated voids, enlarged vacuoles, and large cavities, contrary to the conventional view of a highly ordered organization. We tracked these features longitudinally over weeks and observed the incorporation of new cells during growth. Taken together, non-invasive longitudinal in vivo imaging of lens morphology using AO 2PFM will allow us to directly observe the development or alterations of lens cellular organization in living animals.
RESUMO
Purpose: The mammalian ocular lens is an avascular multicellular organ that grows continuously throughout life. Traditionally, its cellular organization is investigated using dissected lenses, which eliminates in vivo environmental and structural support. Therefore, in vivo optical imaging methods for studying lenses in their native context in live animals are urgently needed. Methods: Here, we demonstrated that two-photon fluorescence microscopy can visualize lens cells in vivo. To maintain subcellular resolution at depth, we used adaptive optics to correct aberrations owing to ocular and lens tissues, which led to substantial signal and resolution improvements. Results: Imaging lens cells up to 980 µm deep, we observed novel cellular organizations including suture-associated voids, enlarged vacuoles, and large cavities, contrary to the conventional view of a highly ordered organization. We tracked these features longitudinally over weeks and observed the incorporation of new cells during growth. Conclusions: Taken together, noninvasive longitudinal in vivo imaging of lens morphology using adaptive optics two-photon fluorescence microscopy will allow us to observe the development or alterations of lens cellular organization in living animals directly.
Assuntos
Cristalino , Animais , Microscopia de Fluorescência , Olho , Células Epiteliais , Fótons , MamíferosRESUMO
The cGMP-phosphodiesterase 6 beta subunit (PDE6B) is an essential component in the phototransduction pathway for light responses in photoreceptor cells. PDE6B gene mutations cause the death of rod photoreceptors, named as hereditary retinitis pigmentosa (RP) in humans and retinal degeneration (RD) in rodents. Here, we report a new RD model, identified from a phenotypic screen of N-ethyl-N-nitrosourea (ENU)-induced mutant mice, which displays retinal degeneration caused by a point mutation in the Pde6b gene that results in PDE6B-T592I mutant protein. The homozygous mutant mice show an extensive loss of rod photoreceptors at the age of 3 weeks; unexpectedly, the loss of rod photoreceptors can be partly rescued by dark rearing. Thus, this RD mutant model displays a light-dependent loss of rod photoreceptors. Both western blot and immunostaining results show very low level of mutant PDE6B-T592I protein in the retina. Structure modeling suggests that the T592I mutation probably affects the function and stability of PDE6B protein by changing intramolecular interactions. We further demonstrate that the expression of wild-type PDE6B delivered by subretinally injected adeno-associated virus (rAAV) prevents photoreceptor cell death in this RD model in vivo. The PDE6B-T592I mutant is, therefore, a valuable RD model for evaluating rAAV-mediated treatment and for investigating the molecular mechanism of light-dependent rod photoreceptor cell death that is related to impaired PDE6B function.
RESUMO
The retina, behind the transparent optics of the eye, is the only neural tissue whose physiology and pathology can be non-invasively probed by optical microscopy. The aberrations intrinsic to the mouse eye, however, prevent high-resolution investigation of retinal structure and function in vivo. Optimizing the design of a two-photon fluorescence microscope (2PFM) and sample preparation procedure, we found that adaptive optics (AO), by measuring and correcting ocular aberrations, is essential for resolving putative synaptic structures and achieving three-dimensional cellular resolution in the mouse retina in vivo. Applying AO-2PFM to longitudinal retinal imaging in transgenic models of retinal pathology, we characterized microvascular lesions with sub-capillary details in a proliferative vascular retinopathy model, and found Lidocaine to effectively suppress retinal ganglion cell hyperactivity in a retinal degeneration model. Tracking structural and functional changes at high-resolution longitudinally, AO-2PFM enables microscopic investigations of retinal pathology and pharmacology for disease diagnosis and treatment in vivo.
Assuntos
Retina , Degeneração Retiniana , Camundongos , Animais , Retina/patologia , Células Ganglionares da Retina , Degeneração Retiniana/patologia , Microscopia de Fluorescência , Óptica e FotônicaRESUMO
Despite the enormous number of studies demonstrating changes in the chaperone-like activity of α-crystallins in vitro, little is known about how these changes influence life-long lens transparency in vivo. Using the γB-crystallin I4F mutant protein as a target for αA-crystallins, we examined how cataract phenotypes are modulated by interactions between α-crystallins with altered chaperone-like activities and γB-I4F proteins in vivo. Double heterozygous α-crystallin knock-out αA(+/-) αB(+/-) mice with a decreased amount of α-crystallins were used to simulate reduced total α-crystallin chaperone-like activity in vivo. We found that triple heterozygous αA(+/-) αB(+/-) γB(I4F/+) mice developed more severe whole cataracts than heterozygous γB(I4F/+) mice. Thus, total chaperone-like activity of α-crystallins is important for maintaining lens transparency. We further tested whether mutant αA-crystallin Y118D proteins with increased chaperone-like activity influenced the whole cataract caused by the γB-I4F mutation. Unexpectedly, compound αA(Y118D/+) γB(I4F/+) mutant lenses displayed severe nuclear cataracts, whereas the lens cortex remained unaffected. Thus, the synergistic effect of αA-Y118D and γB-I4F mutant proteins is detrimental to the transparency only in the lens core. α-Crystallins with different chaperone-like activities are likely required in the lens cortex and nucleus for maintaining transparency.
Assuntos
Catarata , Córtex do Cristalino , Mutação , Cadeia A de alfa-Cristalina , beta-Cristalinas , Substituição de Aminoácidos , Animais , Catarata/genética , Catarata/metabolismo , Catarata/patologia , Linhagem Celular Transformada , Heterozigoto , Córtex do Cristalino/metabolismo , Córtex do Cristalino/patologia , Camundongos , Camundongos Knockout , Cadeia A de alfa-Cristalina/genética , Cadeia A de alfa-Cristalina/metabolismo , beta-Cristalinas/genética , beta-Cristalinas/metabolismoRESUMO
Cataracts, named for pathological light scattering in the lens, are known to be associated with increased large protein aggregates, disrupted protein phase separation, and/or osmotic imbalances in lens cells. We have applied synchrotron phase contrast X-ray micro-computed tomography to directly examine an age-related nuclear cataract model in Cx46 knockout (Cx46KO) mice. High-resolution 3D X-ray tomographic images reveal amorphous spots and strip-like dense matter precipitates in lens cores of all examined Cx46KO mice at different ages. The precipitates are predominantly accumulated in the anterior suture regions of lens cores, and they become longer and dense as mice age. Alizarin red staining data confirms the presence of calcium precipitates in lens cores of all Cx46KO mice. This study indicates that the spatial and temporal calcium precipitation is an age-related event associated with age-related nuclear cataract formation in Cx46KO mice, and further suggests that the loss of Cx46 promotes calcium precipitates in the lens core, which is a new mechanism that likely contributes to the pathological light scattering in this age-related cataract model.
Assuntos
Cálcio/metabolismo , Catarata/metabolismo , Animais , Catarata/patologia , Cristalino/metabolismo , Camundongos , Camundongos Knockout , Microtomografia por Raio-XRESUMO
We have identified a mouse recessive mutation that leads to attenuated and hyperpermeable retinal vessels, recapitulating some pathological features of familial exudative vitreoretinopathy (FEVR) in human patients. DNA sequencing reveals a single nucleotide insertion in the gene encoding the low-density lipoprotein receptor-related protein 5 (LRP5), causing a frame shift and resulting in the replacement of the C-terminal 39 amino acid residues by 20 new amino acids. This change eliminates the last three PPP(S/T)P repeats in the LRP5 cytoplasmic domain that are important for mediating Wnt/beta-catenin signaling. Thus, mutant LRP5 protein is probably unable to mediate its downstream signaling. Immunostaining and three-dimensional reconstructions of retinal vasculature confirm attenuated retinal vessels. Ultrastructural data further reveal that some capillaries lack lumen structure in the mutant retina. We have also verified that LRP5 null mice develop similar alterations in the retinal vasculature. This study provides direct evidence that LRP5 is essential for the development of retinal vasculature, and suggests a novel role played by LRP5 in capillary maturation. LRP5 mutant mice can be a useful model to explore the clinical manifestations of FEVR.
Assuntos
Modelos Animais de Doenças , Mutação da Fase de Leitura , Proteínas Relacionadas a Receptor de LDL/genética , Camundongos , Doenças Retinianas/genética , Vasos Retinianos/anormalidades , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Capilares/anormalidades , Capilares/crescimento & desenvolvimento , Etilnitrosoureia/farmacologia , Éxons , Homozigoto , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Masculino , Camundongos Knockout , Dados de Sequência Molecular , Mutagênese , Mutagênese Insercional , Estrutura Terciária de Proteína , Vasos Retinianos/crescimento & desenvolvimentoRESUMO
Purpose: To investigate the underlying mechanisms for how the mouse Cx50-R205G point mutation, a homologue of the human Cx50-R198W mutation that is linked to cataract-microcornea syndrome, affects proper lens growth and fiber cell differentiation to lead to severe lens phenotypes. Methods: EdU labeling, immunostaining, confocal imaging analysis, and primary lens epithelial cell culture were performed to characterize the lens epithelial cell (LEC) proliferation and fiber cell differentiation in wild-type and Cx50-R205G mutant lenses in vivo and in vitro. Results: The Cx50-R205G mutation severely disrupts the lens size and transparency. Heterozygous and homozygous Cx50-R205G mutant and Cx50 knockout lenses all show decreased central epithelium proliferation while only the homozygous Cx50-R205G mutant lenses display obviously decreased proliferating LECs in the germinative zone of neonatal lenses. Cultured Cx50-R205G lens epithelial cells reveal predominantly reduced Cx50 gap junction staining but no change of the endoplasmic reticulum stress marker BiP. The heterozygous Cx50-R205G lens fibers show moderately disrupted Cx50 and Cx46 gap junctions while the homozygous Cx50-R205G lens fibers have drastically reduced Cx50 and Cx46 gap junctions with severely altered fiber cell shape in vivo. Conclusions: The Cx50-R205G mutation inhibits both central and equatorial lens epithelial cell proliferation to cause small lenses. This mutation also disrupts the assembly and functions of both Cx50 and Cx46 gap junctions in lens fibers to alter fiber cell differentiation and shape to lead to severe lens phenotypes.
Assuntos
Catarata/genética , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Conexinas/genética , Doenças da Córnea/genética , Células Epiteliais/patologia , Cristalino/patologia , Mutação Puntual , Animais , Animais Recém-Nascidos , Catarata/congênito , Catarata/patologia , Células Cultivadas , Doenças da Córnea/patologia , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia de FluorescênciaRESUMO
To test the hypothesis that fast anterograde molecular motor proteins power the slow axonal transport of neurofilaments (NFs), we used homologous recombination to generate mice lacking the neuronal-specific conventional kinesin heavy chain, KIF5A. Because null KIF5A mutants die immediately after birth, a synapsin-promoted Cre-recombinase transgene was used to direct inactivation of KIF5A in neurons postnatally. Three fourths of such mutant mice exhibited seizures and death at around 3 wk of age; the remaining animals survived to 3 mo or longer. In young mutant animals, fast axonal transport appeared to be intact, but NF-H, as well as NF-M and NF-L, accumulated in the cell bodies of peripheral sensory neurons accompanied by a reduction in sensory axon caliber. Older animals also developed age-dependent sensory neuron degeneration, an accumulation of NF subunits in cell bodies and a reduction in axons, loss of large caliber axons, and hind limb paralysis. These data support the hypothesis that a conventional kinesin plays a role in the microtubule-dependent slow axonal transport of at least one cargo, the NF proteins.
Assuntos
Transporte Axonal/genética , Axônios/metabolismo , Encéfalo/metabolismo , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas de Neurofilamentos/metabolismo , Animais , Animais Recém-Nascidos , Axônios/patologia , Encéfalo/anormalidades , Encéfalo/crescimento & desenvolvimento , Gânglios Espinais/anormalidades , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Imuno-Histoquímica , Integrases , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Mutação/genética , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Fibras Nervosas Mielinizadas/metabolismo , Fibras Nervosas Mielinizadas/patologia , Neurônios Aferentes/metabolismo , Neurônios Aferentes/patologia , Nervos Periféricos/anormalidades , Nervos Periféricos/metabolismo , Nervos Periféricos/patologia , Convulsões/genética , Convulsões/metabolismo , Transgenes/genética , Proteínas ViraisRESUMO
PURPOSE: To identify the causative gene mutation for a new dominant cataract in mice and to investigate the molecular basis for how the mutated gene leads to a dense nuclear cataract. METHODS: Genomewide linkage analysis and DNA sequencing were used to determine the gene mutation. Histology, immunohistochemistry, and Western blotting were used to characterize lens phenotypes. Ion concentrations were measured by an inductively coupled plasma-optical emission spectrometer (ICP-OES). RESULTS: A point mutation (A to C) of the gammaB-crystallin gene, which results in the gammaB-S11R mutant protein, was identified in this cataractous mouse line. Homozygous mutant mice developed dense nuclear cataracts associated with disrupted inner lens fiber cells. Immunohistochemistry data revealed gamma-crystallin aggregates at the cell boundaries of inner mature fibers that lose actin filaments. Western blotting showed an increased degradation of crystallin proteins correlated with the nuclear cataract. ICP-OES confirmed a substantial elevation of calcium concentration in mutant lenses. CONCLUSIONS: This dominant cataract was caused by the gammaB-S11R mutation. Mutant gammaB-S11R proteins triggered the gamma-crystallin aggregation that probably disrupted membrane-cytoskeleton structures of inner fiber cells, causing increased calcium influxes. Subsequent activation of calcium-dependent protein degradation and degeneration of inner mature fiber cells led to the dense nuclear cataract.
Assuntos
Catarata/genética , Modelos Animais de Doenças , Núcleo do Cristalino/patologia , Mutação Puntual , gama-Cristalinas/genética , Animais , Western Blotting , Cálcio/metabolismo , Catarata/metabolismo , Mapeamento Cromossômico , Cristalinas/metabolismo , Genes Dominantes , Núcleo do Cristalino/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia Confocal , Fenótipo , Análise de Sequência de DNA , Espectrometria por Raios XRESUMO
PURPOSE: To understand the underlying molecular mechanism for a dominant cataract caused by a point mutation in the gammaD-crystallin gene. METHODS: A dominant cataractous mouse line was identified from chemically induced mouse mutations by phenotypic screening with slit lamp examination. Genomewide linkage analysis and DNA sequencing were used to determine the causative gene mutation. Histology, immunohistochemistry, Western blotting, and in vitro transfection studies were used to characterize mutant lenses. RESULTS: Cataracts in mutant mice were caused by a point mutation in the gammaD-crystallin gene (gammaD-V76D). Intranuclear gamma-crystallin aggregates, incomplete denucleation, and decreased connexins were observed in mutant lens fiber cells. Mutant gammaD-V76D proteins became less soluble in the lens, and structural modeling suggested that the substituted aspartic acid residue (D) altered hydrogen bond formation and surface electrostatic potential of the protein. Unexpectedly, the formation of cold cataracts, which occurred in wild-type lenses at low temperature, was abolished in gammaD-V76D mutant lenses. In vitro transfection studies revealed that wild-type gammaD proteins were uniformly distributed in the cytosol and nucleus of transfected cells, whereas gammaD-V76D proteins formed cytosolic and nuclear aggregates. CONCLUSIONS: Mutant gammaD-V76D reduces protein solubility in the lens and forms substantial intranuclear aggregates that disrupt the denucleation process of inner lens fiber cells. Sustained fiber cell nuclei and nuclear remnants scatter light, whereas other downstream events, such as decreased connexins, presumably disrupt gap junction communication and lens homeostasis, further contributing to the cataract phenotype in mutant lenses. This work also suggests that gammaD-crystallin is one of the crucial components for the formation of cold cataracts in vivo.
Assuntos
Catarata/metabolismo , Células Epiteliais/metabolismo , Cristalino/metabolismo , gama-Cristalinas/metabolismo , Substituição de Aminoácidos , Animais , Catarata/etiologia , Catarata/genética , Linhagem Celular Transformada , Núcleo Celular/metabolismo , Temperatura Baixa/efeitos adversos , Conexinas/metabolismo , Citosol/metabolismo , Células Epiteliais/citologia , Feminino , Cristalino/citologia , Masculino , Camundongos , Camundongos Mutantes , Fenótipo , Mutação Puntual , Estrutura Terciária de Proteína , Ruptura , Solubilidade , Água/metabolismo , gama-Cristalinas/química , gama-Cristalinas/genéticaRESUMO
Neonatal cataracts remain the most common cause of visual loss in children worldwide and have diverse, often unknown, etiologies. This review summarizes current knowledge about the detection, treatment, genetics, risk factors, and molecular mechanisms of congenital cataracts. We emphasize significant progress and topics requiring further study in both clinical cataract therapy and basic lens research. Advances in genetic screening and surgical technologies have improved the diagnosis, management, and visual outcomes of affected children. For example, mutations in lens crystallins and membrane/cytoskeletal components that commonly underlie genetically inherited cataracts are now known. However, many questions still remain regarding the causes, progression, and pathology of neonatal cataracts. Further investigations are also required to improve diagnostic criteria for determining the timing of appropriate interventions, such as the implantation of intraocular lenses and postoperative management strategies, to ensure safety and predictable visual outcomes for children. Birth Defects Research 109:734-743, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Catarata/genética , Catarata/terapia , Catarata/congênito , Catarata/epidemiologia , Extração de Catarata/efeitos adversos , Oftalmopatias Hereditárias/genética , Predisposição Genética para Doença/genética , Testes Genéticos/métodos , Humanos , Implante de Lente Intraocular/efeitos adversos , Cristalino/patologia , Lentes Intraoculares/efeitos adversos , Fatores de Risco , Acuidade VisualRESUMO
Transforming growth factor ß (TGFß) and fibroblast growth factor (FGF) signaling pathways play important roles in the proliferation and differentiation of lens epithelial cells (LECs) during development. Low dosage bFGF promotes cell proliferation while high dosage induces differentiation. TGFß signaling regulates LEC proliferation and differentiation as well, but also promotes epithelial-mesenchymal transitions that lead to cataracts. Thus far, it has been difficult to recapitulate the features of germinative LECs in vitro. Here, we have established a LEC culture protocol that uses SB431542 (SB) compound to inhibit TGFß/Smad activation, and found that SB treatment promoted mouse LEC proliferation, maintained LECs' morphology and distinct markers including N-cadherin, c-Maf, Prox1, and αA-, αB-, and ß-crystallins. In contrast, low-dosage bFGF was unable to sustain those markers and, combined with SB, altered LECs' morphology and ß-crystallin expression. We further found that Matrigel substrate coatings greatly increased cell proliferation and uniquely affected ß-crystallin expression. Cultured LECs retained the ability to differentiate into γ-crystallin-positive lentoids by high-dosage bFGF treatment. Thus, a suppression of TGFß/Smad signaling in vitro is critical to maintaining characteristic features of mouse LECs, especially expression of the key transcription factors c-Maf and Prox1.
Assuntos
Diferenciação Celular , Células Epiteliais/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Cristalino/citologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Benzamidas/farmacologia , Biomarcadores , Proliferação de Células , Células Cultivadas , Dioxóis/farmacologia , Células Epiteliais/efeitos dos fármacos , Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-maf/genética , Proteínas Proto-Oncogênicas c-maf/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
In the current study we describe the changes of overall organization of lens fiber cells in connexin 46 (Cx46) and connexin 50 (Cx50) knockout mice. Morphometric analyses and the application of immunocytochemical techniques revealed that in Cx46 knockout lens (Cx46 -/-), where Cx50 is expressed alone, the postnatal differentiation of secondary fiber cells proceeds faster and is characterized by an increased number of smaller fiber cells. Conversely, in Cx50 knockout mice (Cx50 -/-), the lenticular mass is considerably reduced and characterized by a small number of fiber cells added during the postnatal period. The process of terminal differentiation was impaired and generated larger fiber cells still possessing cytoplasmic organelles. Freeze-fracture and fracture labeling revealed that the junctional assembly, packing organization and topographic interactions between connexons and MP26 differed when Cx46 and Cx50 were co-assembled in the wild-type or expressed separately in the two distinct knockout phenotypes. Filipin cytochemistry provided indirect evidence that Cx46 and Cx50 expressed alone are recruited into different lipid environments. Our results represent the structural proof that interaction of connexins and MP26 contributes to the overall organization of the fiber cells.