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.
Highlights
Retinitis pigmentosa (RP) is one form of inherited retinal degenerative disease (IRD) causing incurable blindness
Retinitis pigmentosa (RP) is an inherited form of retinal degeneration characterized by primary rod photoreceptor cell death followed by cone loss
While a high number of different mutations can be detrimental to rod photoreceptors, some common molecular events characterize the progression of photoreceptor cell death and among these is the increase in cyclic guanosine monophosphate levels and in calcium influx [3,4,5,6]
Summary
Retinitis pigmentosa (RP) is one form of inherited retinal degenerative disease (IRD) causing incurable blindness. Therapeutic approaches for the preservation of diseased photoreceptor cells face several challenges such as the high genetic heterogeneity [2], the impossibility of collecting biopsies from RP patients, the presence of a blood–retina barrier that hinders drug delivery [7] and the lack of an appropriate in vitro model for drug screening. This last issue relates to the fact that photoreceptor cells are postmitotic neurons and cannot regenerate themselves; cell lines cannot be generated from adult photoreceptor cells. All these problems make the study of rod photoreceptors that undergo cell death in RP demanding
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