RESUMO
Purpose: Purpose The role of endothelial Yes-associated protein 1 (YAP) in the pathogenesis of retinal angiogenesis and the astrocyte network in the mouse oxygen-induced retinopathy (OIR) model is unknown. Methods: For in vivo studies, OIR was induced in conditional endothelial YAP knockout mice and their wild-type littermates. Retinal vascularization and the astrocyte network were evaluated by whole-mount fluorescence and Western blotting. In vitro experiments were performed in astrocytes cultured with human microvascular endothelial cell-1-conditioned medium to analyze the mechanisms underlying the effect of endothelial YAP on astrocytes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Astrócitos/patologia , Proteínas de Ciclo Celular/fisiologia , Células Endoteliais/metabolismo , Fator Inibidor de Leucemia/metabolismo , Neovascularização Retiniana/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Proliferação de Células , Células Cultivadas , Meios de Cultivo Condicionados , Citoplasma/metabolismo , Modelos Animais de Doenças , Técnica Indireta de Fluorescência para Anticorpo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Oxigênio/toxicidade , Neovascularização Retiniana/induzido quimicamente , Neovascularização Retiniana/patologia , Vasos Retinianos/citologia , Retinopatia da Prematuridade/induzido quimicamente , Retinopatia da Prematuridade/metabolismo , Retinopatia da Prematuridade/patologia , Proteínas de Sinalização YAPRESUMO
PURPOSE: Vacuolar protein sorting 35 (Vps35) mutations and protein dysfunction have been linked to the hyperphosphorylation and accumulation of tau protein in a number of central neurodegenerative disorders. The aims of the present study were to investigate the mechanism underlying the tau hyperphosphorylation caused by Vps35 deficiency. METHODS: The cells used in this study were primary retinal ganglion cells (RGCs). The rat retinal glutamate excitotoxicity model was used in vivo. Fresh retinal tissues or eyeballs were collected at different time points. The expression and interactions of Vps35, Cdk5/p35, tau hyperphosphorylation, LAMP1, EEA1 and UBE1 in RGCs were studied by immunofluorescence staining, Western blotting, and immunoprecipitation. RESULTS: The downregulation and overexpression of Vps35 increased and decreased the expression of p35 and tau hyperphosphorylation, respectively. More important, roscovitine, a Cdk5 inhibitor, could effectively decrease the hyperphosphorylated tau level induced by Vps35 deficiency. Furthermore, this study confirmed that the inhibition of Vps35 could increase the activity of Cdk5/p35 by affecting the lysosomal degradation of p35 and lead to the degeneration of RGCs. CONCLUSIONS: These findings demonstrate the possibility that Cdk5/p35 acts as a "cargo" of Vps35 and provide new insights into the pathogenesis of RGC degeneration caused by hyperphosphorylated tau protein. Vps35 is a potential target for basic research and clinical treatment of RGC degeneration in many ocular diseases such as glaucoma.
Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Fosfotransferases/metabolismo , Células Ganglionares da Retina/metabolismo , Proteínas de Transporte Vesicular/deficiência , Proteínas tau/metabolismo , Animais , Western Blotting , Células Cultivadas , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Regulação para Baixo , Técnica Indireta de Fluorescência para Anticorpo , Ácido Glutâmico/toxicidade , Proteínas de Membrana Lisossomal/metabolismo , Masculino , Camundongos , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/metabolismo , Roscovitina/farmacologia , Transfecção , Enzimas Ativadoras de Ubiquitina/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
OBJECTIVES: To testify that endothelial cells (ECs) induce astrocyte maturation by leukaemia inhibitory factor (LIF) secretion. MATERIALS AND METHODS: In vivo experiments, mice bearing floxed alleles of YAP were crossed with mice expressing a Cre recombinase driven by the endothelial Tek promoter (Tek-Cre) to finally obtain the following three genotypes: YAPf/f , Tek-Cre; YAPf/w , Tek-Cre; and YAPf/f . Retinal vascularization and astrocyte network were evaluated by whole-mount fluorescence and Western blotting. In vitro, experiments were performed in an astrocyte and human microvascular endothelial cell (HMEC-1) coculture model to analyse the mechanisms underlying the effect of endothelial YAP on astrocytes. RESULTS: In vivo, YAPf/f ;Tek-Cre mice showed delayed angiogenesis, sparse vessels and decreased glial fibrillary acidic protein (GFAP)+ astrocytes but aberrant growth of endothelial networks and immature astrocytes (platelet-derived growth factor A, PDGFRA+ astrocytes) overgrowth. In vitro, Yap deletion attenuated the LIF release that delayed the maturation of retinal astrocyte which was consistent with the results of HMEC-1-astrocyte coculture. The effect of YAP overexpression on LIF-LIFR axis in HMEC-1 interferes the GFAP expression of astrocyte. In contrast, LIF protein rescues the astrocytic GFAP expression when EC YAP was inhibited by siRNAs. CONCLUSIONS: We show that EC yes-associated protein (YAP) is not only a critical coactivator of Hippo signalling in retinal vessel development but also plays an essential role in retinal astrocyte maturation by regulating LIF production.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Astrócitos/metabolismo , Fator Inibidor de Leucemia/metabolismo , Retina/metabolismo , Vasos Retinianos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Astrócitos/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Técnicas de Cocultura/métodos , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neovascularização Fisiológica/fisiologia , Neurogênese/fisiologia , Retina/fisiologia , Vasos Retinianos/fisiologia , Proteínas de Sinalização YAPRESUMO
AIM: To explore the more suitable concentration of glutamate or N-methyl-D-aspartic acid (NMDA) for intravitreal injection to establish a rat model of retinal neurodegeneration. METHODS: We injected different doses of glutamate (20 or 50 nmol) or NMDA (40 nmol) into the vitreous chambers of rats, then measured the concentration of glutamate and retinal thickness, quantified apoptotic cells and determined the degree of tau hyperphosphorylation at different time points. T-test was used for comparison of two groups. One-way ANOVA and Turkey's multiple comparisons test were used for comparisons of different groups, and P values below 0.05 were considered statistically significant. RESULTS: The glutamate level in the rats treated with 50 nmol of glutamate was twice that of the control group and persisted two weeks. Seven days after intravitreal injection of 50 nmol of glutamate, three parameters [inner retinal thickness (IRT), retinal thickness (RT) and ganglion cell layer (GCL) cell number] were reduced significantly. Furthermore, numerous TUNEL-positive cells were observed in the GCL one day after intravitreal injection of 50 nmol of glutamate, the expression of the apoptosis-related factor cleaved casepase-3 was markedly increased compared with the expression levels in the other treatment groups, and the expression levels of tau s396 and tau s404 were significantly increased compared with those in the control group. CONCLUSION: This study demonstrates that the intravitreal injection of 50 nmol of glutamate can establish the more effective retinal neurodegeneration animal model relative to other treatment groups.