Investigating the molecular mechanisms of Late-Onset Retinal Degeneration using patient-specific induced pluripotent stem cells

Abstract

Purpose : Late-Onset Retinal Degeneration (L-ORD) is a rare autosomal dominant disorder that shares clinical similarities to more prevalent retinal degenerations, such as age-related macular degeneration. L-ORD is caused by a single S163R amino acid substitution in the globular domain of the CTRP5 protein. The CTRP5 gene is contained within the 3’-untranslated region of another gene, which encodes the membrane-frizzled related protein (MFRP). Investigating this genetically simple disease will provide us with valuable insight into the shared mechanisms of other retinal degenerative diseases and help us identify novel targets for therapy.

Methods : We generated induced pluripotent stem cells (iPSCs) from fibroblasts of two L-ORD-affected and two unaffected siblings. iPSCs were characterized by the expression of pluripotent markers: SSEA4, SOX2, OCT4, and NANOG. Karyotype analysis revealed no aberrations. Sequencing confirmed that the patients’ cells retained the S163R point mutation characteristic of L-ORD. iPSCs were differentiated into RPE using a directed differentiation protocol. CTRP5 and MFRP expression levels were assessed by qPCR, Western blot, and immunostaining. Intracellular calcium levels were measured with ratiometric calcium dye, Fura-2.

Results : RPE differentiated from iPSCs of L-ORD patients and their healthy sibling express similar amounts of CTRP5 protein. mRNA expression of MFRP is also not significantly different between patients and healthy cells. However, patient’s cells show characteristic deposits that are reminiscent of deposits seen in the eyes of L-ORD patients. Calcium imaging results demonstrate lower baseline intracellular calcium levels in patient cells as compared to their healthy siblings.

Conclusions : L-ORD patient RPE cells show cellular endophenotypes that are reminiscent of phenotype seen in the eyes of L-ORD patients. Reduced baseline calcium in L-ORD patient cells suggests defective calcium homeostasis that might contribute to accumulation of intra and sub-cellular deposits.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.