Transcriptome profiling of NIH3T3 cell lines expressing opsin and the P23H opsin mutant identifies candidate drugs for the treatment of retinitis pigmentosa

Abstract

Mammalian cells are commonly employed in screening assays to identify active compounds that could potentially affect the progression of different human diseases including retinitis pigmentosa (RP), a class of inherited diseases causing retinal degeneration with compromised vision. Using transcriptome analysis, we compared NIH3T3 cells expressing wildtype (WT) rod opsin with a retinal disease-causing single P23H mutation. Surprisingly, heterologous expression of WT opsin in NIH3T3 cells caused more than a 2-fold change in 783 out of 16,888 protein coding transcripts. The perturbed genes encoded extracellular matrix proteins, growth factors, cytoskeleton proteins, glycoproteins and metalloproteases involved in cell adhesion, morphology and migration. A different set of 347 transcripts was either up- or down-regulated when the P23H mutant opsin was expressed suggesting an altered molecular perturbation compared to WT opsin. Transcriptome perturbations elicited by drug candidates aimed at mitigating the effects of the mutant protein revealed that different drugs targeted distinct molecular pathways that resulted in a similar phenotype selected by a cell-based high-throughput screen. Thus, transcriptome profiling can provide essential information about the therapeutic potential of a candidate drug to restore normal gene expression in pathological conditions.

A novel small molecule chaperone of rod opsin and its potential therapy for retinal degeneration

“…Scriptaid, a pan-histone deacetylase inhibitor, also showed strong activity in rescuing P23H opsin transport (Supplementary Data 1 ). Moreover, co-treatment with YC-001 and scriptaid produced a synergistic effect, suggesting distinctive targets for these two active compounds 33 . YC-001 did not affect the amount or stability of the clarin-1 N48K mutant that causes Type III Usher syndrome 34 in HEK 293 cells, suggesting the activity of YC-001 is specific to rod opsin (Supplementary Fig.…”

“…3d ). The glycosylation of opsin varies between cell types in which it is expressed 13 , 19 , 33 . Here we used NIH3T3 cells to show that immature glycosylated P23H rod opsin can be further processed in cells treated with 9- cis -retinal or YC-001.…”

“…In contrast, for the other active compound scriptaid that also increases the transport of P23H opsin to the plasma membrane, there is no evidence to show that it binds to rod opsin. First, scriptaid does not affect the fluorescence of Trp 265 in rod opsin; second, scriptaid does not compete with 9- cis -retinal for isorhodopsin regeneration; third, scriptaid does not affect the glycosylation of P23H opsin; and finally, the synergy between scriptaid and YC-001 on the transport of P23H opsin suggests different mechanisms of actions between the two molecules 33 . In our previous report, scriptaid as a histone deacetylase inhibitor, affected a total of 6% of the entire transcriptome 33 .…”

“…First, scriptaid does not affect the fluorescence of Trp 265 in rod opsin; second, scriptaid does not compete with 9- cis -retinal for isorhodopsin regeneration; third, scriptaid does not affect the glycosylation of P23H opsin; and finally, the synergy between scriptaid and YC-001 on the transport of P23H opsin suggests different mechanisms of actions between the two molecules 33 . In our previous report, scriptaid as a histone deacetylase inhibitor, affected a total of 6% of the entire transcriptome 33 . Rather than directly binding to rod opsin, scriptaid may improve P23H opsin transport through transcriptional regulation of multiple pathways including cytoskeleton dynamics, proteolysis, and vesicle transport.…”

“…Three NIH3T3 stable cell lines, NIH3T3-(WT-opsin/GFP), NIH3T3-(P23H opsin/GFP), and NIH3T3-(GFP) were previously generated which stably express mouse WT-opsin and GFP; P23H opsin and GFP; and only GFP, respectively 18 , 33 . GFP is expressed separately from opsin in these cell lines.…”