An easy, rapid method to isolate RPE cell protein from the mouse eye

Wei, Hong; Xun, Zixian; Granado, Herta; Wu, Angela Ruohao; Handa, James T.

doi:10.1016/j.exer.2015.09.015

Cited by 44 publications

(34 citation statements)

References 8 publications

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“…Samples were then spun at 13,300 RPM at 4°C for 15 min, and the supernatant was normalized for loading by BCA assay to 20 μg/tissue. RPE protein lysate was prepared according to a described protocol ( Wei et al, 2016 ).…”

Section: Methodsmentioning

confidence: 99%

Biochemical adaptations of the retina and retinal pigment epithelium support a metabolic ecosystem in the vertebrate eye

Kanow

Giarmarco

Jankowski

et al. 2017

eLife

295

258

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Here we report multiple lines of evidence for a comprehensive model of energy metabolism in the vertebrate eye. Metabolic flux, locations of key enzymes, and our finding that glucose enters mouse and zebrafish retinas mostly through photoreceptors support a conceptually new model for retinal metabolism. In this model, glucose from the choroidal blood passes through the retinal pigment epithelium to the retina where photoreceptors convert it to lactate. Photoreceptors then export the lactate as fuel for the retinal pigment epithelium and for neighboring Müller glial cells. We used human retinal epithelial cells to show that lactate can suppress consumption of glucose by the retinal pigment epithelium. Suppression of glucose consumption in the retinal pigment epithelium can increase the amount of glucose that reaches the retina. This framework for understanding metabolic relationships in the vertebrate retina provides new insights into the underlying causes of retinal disease and age-related vision loss.

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Section: Methodsmentioning

confidence: 99%

Biochemical adaptations of the retina and retinal pigment epithelium support a metabolic ecosystem in the vertebrate eye

Kanow

Giarmarco

Jankowski

et al. 2017

eLife

295

258

View full text Add to dashboard Cite

show abstract

“…Western Blotting. Purified retina and RPE protein lysates were obtained using an established protocol (78). For RPE, eyes from three to five eyes were pooled, constituting one independent observation.…”

Section: Methodsmentioning

confidence: 99%

Chronic Dicer1 deficiency promotes atrophic and neovascular outer retinal pathologies in mice

Wright

Uehara

Kim

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Degeneration of the retinal pigmented epithelium (RPE) and aberrant blood vessel growth in the eye are advanced-stage processes in blinding diseases such as age-related macular degeneration (AMD), which affect hundreds of millions of people worldwide. Loss of the RNase DICER1, an essential factor in micro-RNA biogenesis, is implicated in RPE atrophy. However, the functional implications of DICER1 loss in choroidal and retinal neovascularization are unknown. Here, we report that two independent hypomorphic mouse strains, as well as a separate model of postnatal RPE-specific DICER1 ablation, all presented with spontaneous RPE degeneration and choroidal and retinal neovascularization. DICER1 hypomorphic mice lacking critical inflammasome components or the innate immune adaptor MyD88 developed less severe RPE atrophy and pathological neovascularization. DICER1 abundance was also reduced in retinas of the JR5558 mouse model of spontaneous choroidal neovascularization. Finally, adenoassociated vector-mediated gene delivery of a truncated DICER1 variant (OptiDicer) reduced spontaneous choroidal neovascularization in JR5558 mice. Collectively, these findings significantly expand the repertoire of DICER1 in preserving retinal homeostasis by preventing both RPE degeneration and pathological neovascularization.

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“…Samples were then spun at 13,300 RPM at 4°C for 15 min, and the supernatant was normalized for loading by BCA assay to 20 mg/tissue. RPE protein lysate was prepared according to a described protocol (51).…”

Section: Tissue Preparations For Immunoblottingmentioning

confidence: 99%

Biochemical adaptations of the retina and retinal pigment epithelium support a metabolic ecosystem in the vertebrate eye

Kanow

Giarmarco

Jankowski

et al. 2017

Preprint

120

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1Here we report multiple lines of evidence for a comprehensive model for retinal 2 energy metabolism. Metabolic flux, locations of key enzymes and our finding that 3 glucose enters the neural retina almost entirely through photoreceptors support a 4 conceptually new model for retinal metabolism. In this model, glucose from the 5 choroidal blood supply passes through the retinal pigment epithelium to the retina 6 where photoreceptors convert it to lactate. Photoreceptors then export the lactate 7 as fuel for the retinal pigment epithelium and for neighboring Müller glial cells. A 8 key feature of this model is that aerobic glycolysis in photoreceptors produces 9 lactate to suppress glycolysis in the neighboring retinal pigment epithelium. That 10 enhances the flow of glucose to the retina by minimizing consumption of glucose 11 within the retinal pigment epithelium. This framework for metabolic relationships 12 in retina provides new insights into the underlying causes of retinal disease, age-13 related vision loss and metabolism-based therapies.

show abstract

An easy, rapid method to isolate RPE cell protein from the mouse eye

Cited by 44 publications

References 8 publications

Biochemical adaptations of the retina and retinal pigment epithelium support a metabolic ecosystem in the vertebrate eye

Biochemical adaptations of the retina and retinal pigment epithelium support a metabolic ecosystem in the vertebrate eye

Chronic Dicer1 deficiency promotes atrophic and neovascular outer retinal pathologies in mice

Biochemical adaptations of the retina and retinal pigment epithelium support a metabolic ecosystem in the vertebrate eye

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