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New In Vitro Cellular Model for Molecular Studies of Retinitis Pigmentosa.

ABSTRACT: Retinitis pigmentosa (RP) is an inherited form of retinal degeneration characterized by primary rod photoreceptor cell death followed by cone loss. Mutations in several genes linked to the disease cause increased levels of cyclic guanosine monophosphate (cGMP) and calcium ion influxes. The purpose of this project was to develop a new in vitro photoreceptor degeneration model for molecular studies of RP. 661W cells were genetically modified to stably express the neural retina leucine zipper (NRL) transcription factor. One clone (661W-A11) was selected based on the expression of Nrl target genes. 661W-A11 showed a significant increase in expression of rod-specific genes but not of cone-specific genes, compared with 661W cells. Zaprinast was used to inhibit phosphodiesterase 6 (PDE6) activity to mimic photoreceptor degeneration in vitro. The activation of cell death pathways resulting from PDE6 inhibition was confirmed by detection of decreased viability and increased intracellular cGMP and calcium, as well as activation of protein kinase G (PKG) and calpains. In this new in vitro system, we validated the effects of previously published neuroprotective drugs. The 661W-A11 cells may serve as a new model for molecular studies of RP and for high-throughput drug screening.

SUBMITTER: Huang L

PROVIDER: S-EPMC8235468 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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New In Vitro Cellular Model for Molecular Studies of Retinitis Pigmentosa.

Huang Li L Kutluer Meltem M Adani Elisa E Comitato Antonella A Marigo Valeria V

International journal of molecular sciences 20210616 12

Retinitis pigmentosa (RP) is an inherited form of retinal degeneration characterized by primary rod photoreceptor cell death followed by cone loss. Mutations in several genes linked to the disease cause increased levels of cyclic guanosine monophosphate (cGMP) and calcium ion influxes. The purpose of this project was to develop a new in vitro photoreceptor degeneration model for molecular studies of RP. 661W cells were genetically modified to stably express the neural retina leucine zipper (NRL) ...[more]

PMID: 34208617

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Project description:PurposeRetinitis pigmentosa (RP) is the most common cause of inherited blindness, with onset occurring as early as 4 years of age in certain rare but severe forms caused by mutations in the gamma subunit of phosphodiesterase 6 (PDE6). Studies in humans and mice have shown that RP pathology begins with progressive photoreceptor death, which then drives changes in downstream neurons, neighboring retinal pigment epithelium (RPE), and vasculature. Here, we present the first detailed analysis of RP disease progression in Pde6g-deficient mice.DesignExperimental study of an RP mouse model.SubjectsWe studied Pde6g-/- and Pde6g+/- mice at the age of 7, 16, 30, 44, and 56 days with n = 2 to 5 per group and time point.MethodsPhotoreceptor degeneration and retinal remodeling were analyzed in retinal sections by immunofluorescence. Retinal blood vessel degradation was analyzed in flat-mounted retinas immunolabeled for isolectin GS-IB4. Protein expression was measured by immunoblot. Acellular capillaries were assessed in trypsin-digested and hematoxylin-eosin-stained retinas at postnatal day (P) 44. Retinal pigment epithelium cells were delineated in flat-mounted RPE-choroid-sclera by immunolabeling for the cell-adhesion protein β-catenin.Main outcome measuresImmunofluorescence and morphometry (quantitative analysis of outer nuclear layer, dendrite area, vessel area, acellular vessels, RPE cell size, number of nuclei per RPE cell, RPE cell eccentricity, and RPE cell solidity).ResultsThis novel RP model exhibits early onset and rapid rod degeneration, with the vast majority gone by P16. This pathology leads to retinal remodeling, including changes of inner retinal neurons, early activation of glia cells, degradation of retinal vasculature, and structural abnormalities of the RPE.ConclusionsThe pathology in our Pde6g-/- mouse model precisely mirrors human RP progression. The results demonstrate the significant role of the gamma subunit in maintaining phosphodiesterase activity and provide new insights into the disease progression due to Pde6g deficiency.Financial disclosuresProprietary or commercial disclosure may be found after the references.

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New In Vitro Cellular Model for Molecular Studies of Retinitis Pigmentosa.

Publications

New In Vitro Cellular Model for Molecular Studies of Retinitis Pigmentosa.

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