Phd Student Johannes Kepler University Linz, Medical Faculty Linz, Oberosterreich, Austria
Abstract: Retinitis Pigmentosa (RP), caused by mutations in PRPF31, leads to progressive photoreceptor loss and blindness. This study integrates human-induced pluripotent stem cell (hiPSC)-derived retinal organoids with single-cell RNA sequencing (scRNASeq) to uncover transcriptomic changes and molecular mechanisms underlying RP. Patient-specific iPSCs with a PRPF31 mutation (c.1115_1125 del11) and a control line were differentiated into retinal organoids (ROs) mimicking in vivo development and analyzed at early (d:day85) and late (d285) developmental stages. scRNASeq profiled transcriptomic alterations across retinal populations, including photoreceptors, ganglion cells, and Müller glia. Differentially expressed genes (DEGs) were identified and subjected to pathway enrichment analysis. Key findings were validated through RT-qPCR, immunofluorescence (IF), and functional assessment by multielectrode array (MEA) recordings. In PRPF31-mutant ROs, photoreceptors showed early dysfunction with downregulation of phototransduction and oxidative phosphorylation pathways, progressing to degeneration by d285. Müller glia exhibited upregulation of gliosis and inflammatory markers, while ganglion cells displayed disrupted axon guidance and neuronal differentiation pathways. Temporal comparisons highlighted early photoreceptor dysfunction and late-stage degeneration, linking these changes to aging-related disease processes. Pathway enrichment implicated neurodegenerative and mitochondrial dysfunction as central drivers of RP. Validation through RT-qPCR and IF confirmed those findings. Functional MEA recordings demonstrated diminished electrophysiological activity in PRPF31 ROs. This study shows that PRPF31-mutant ROs recapitulate RP progression, linking late-stage degeneration to aging-related processes. Mitochondrial dysfunction and chronic neuroinflammation emerge as key drivers, offering insights for targeted therapies to preserve photoreceptors and reduce gliosis.
