Abstract
Purpose :
Retinitis Pigmentosa (RP) is an inherited degenerative disease characterized by photoreceptor cell death. Several studies, mostly in Pde6b mutant retinas, identified the mechanisms of rod photoreceptor cell death involving excess of intracellular calcium and cGMP, activation of calpain and PKG and nuclear translocation of AIF. One of the limits that hamper the development of novel therapeutic strategies resides on the lack of a reliable in vitro system for high-throughput drug screenings. The purpose of this study was to generate a genetically modified cell line, derived from 661W photoreceptor progenitor cells, able to model the disease in vitro.
Methods :
661W cells were genetically modified to stably express the neural retina leucine zipper (NRL) rod specific transcription factor. qPCR and immunofluorescence were used to select a clone, highly expressing rod specific genes. Cell differentiation was achieved with a culture medium containing retinoic acid (RA), basic fibroblast growth factor (bFGF), taurine and sodium butyrate. A stress protocol for mimicking photoreceptor degeneration was based on the the PDE6 inhibitor zaprinast.
Results :
661W cells were infected with a retrovirus expressing the NRL transcription factor. Out of 14 cell clones, one was selected based on expression of Nrl and Cnga1 genes. qPCR analyzed rod-specific gene expression, such as Rho, Pde6b, Cngb1, Gnat, Guca, Crx, and showed significant higher levels in this clone compared to the 661W cell line. Cone-specific genes, otherwise, did not change level of expression. We developed a differentiation protocol based on FBS withdrawal and exposure to bFGF, RA, Taurine and sodium butyrate. After differentiation, based on qPCR and immunofluorescence, cells showed an elongated shape and augmented expression of rod specific genes, when compared to not differentiated cells. To mimic the degeneration process caused by increased cGMP, we exposed the cells to zaprinast, an inhibitor of PDE6, and found an increased cell death accompanied by increased intracellular calcium and calpain activation. The effects of known, previously published, neuroprotective agents were tested in the new in vitro model.
Conclusions :
The new in vitro model for rod photoreceptor degeneration will be instrumental for high-throughput screenings and testing of new compounds that can act as novel therapeutic approaches for this blinding disease.
This is a 2021 ARVO Annual Meeting abstract.