The study of disease onset and progression in human is limited by the fact that patients are often symptomatic only at the end stage disease and there is limited access to patient material. iPS cell technology provides a possibility to develop in vitro disease models using patient-specific iPS cells, study disease onset and progression, and identify potential therapeutic intervention. We have focused our attention on Late-Onset Retinal Degeneration (L-ORD), which is caused by a dominant point mutation in a bicistronic gene that codes for two proteins MFRP and CTRP5. The mutation changes one base in the 3-UTR of MFRP and S163R amino acid substitution in the 281 amino acids long CTRP5 protein. We have generated and characterized karyotypically normal four iPS cell clones from two L-ORD patients and five from their two unaffected siblings. All of the patient lines carry the L-ORD mutation and control lines carry the wild type sequence. All iPS cell lines were differentiated into RPE at the same passage and RPE was grown as polarized monolayers on semipermeable transwell membranes for analysis. Our preliminary analysis shows that L-ORD RPE consistently secrete less CTRP5 in the apical culture medium as compared to healthy cells. Recent literature evidence suggests that the mutation does not affect secondary protein folding but affects tertiary structure of CTRP5 oligomers. Because CTRP5 expression is unchanged at the RNA level in mutant cells, it led us to hypothesize that the mutant protein oligomers aggregate in the ER and lead to reduced CTRP5 secretion in the apical medium. This will lead to an up-regulation of ER Unfolded Protein Response (UPR) in patient derived cells. In addition, we have developed methods to culture iPS cell derived RPE cells in 96-well and 384-well microtiter plates to optimize high throughput screening assays. RPE cells were transferred to 384-well plates at committed and immature RPE stages. Our results show that these cells continue to differentiate and mature in these plates and attain RPE phenotype. We have also successfully optimized a multiplex gene expression assay that monitors the differentiation stage of iPS cell derived RPE cells (published in 2014). We have optimized a high throughput screen for proteotoxic stress induced RPE cell death rescue. Using the commercially available LOPAC compound library we identified eight different compounds that can rescue RPE cell death caused by proteotoxicity. We are in the process of validating these drugs and characterizing the signaling pathways that are affected by these drugs and are important for RPE biology. These compounds will lead to potential therapeutic drugs for retinal degenerative diseases.
